Multifunctional protease inhibitors and their use in treatment of disease

ABSTRACT

Fusion proteins of protease inhibitors are provided, in particular fusion proteins of alpha 1-antitrypsin (AAT) and a second protease inhibitor, such as secretory leukocyte protease inhibitor (SLPI) or tissue inhibitor of metalloproteases (TIMP). Polynucleotides encoding the fusion proteins, vectors comprising such polynucleotides, and host cells containing such vectors are also provided. Methods of making the fusion proteins of the invention are also provide, as well as methods of using the fusion proteins, for example to inhibit protease activity in a biological sample or in the treatment of an individual suffering from, or at risk for, a disease or disorder involving unwanted protease activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the provisional patentapplication U.S. Ser. No. 60/256,699, filed Dec. 18, 2000, andprovisional patent application Serial No. 60/331,966, filed Nov. 20,2001 which are in addition hereby incorporated by reference in theirentirety.

TECHNICAL FIELD

[0002] This invention relates to fusion proteins of protease inhibitors,and to methods of making and using these fusion proteins, pharmaceuticalcompositions and kits comprising these fusion proteins, and topolynucleotides encoding these fusion proteins.

BACKGROUND OF THE INVENTION

[0003] Protease/protease inhibitor imbalances are a common feature ofchronic diseases of humans. Examples of diseases and pathologicalconditions in which an imbalance of proteases and their inhibitors isimplicated include rheumatoid and other forms of arthritis, tumormetastasis, tumor angiogenesis, periodontal disease, corneal, epidermal,and gastric ulceration, osteoporosis, Paget's disease of bone,glomerulonephritis, atopic dermatitis, psoriasis, scleroderma, pressureatrophy of bone or tissues, cholesteatoma, nerve cell disorders, organinjury due to ischemia-reperfusion (including local sequelae ofmyocardial anoxia), malaria, chronic wound healing, Chagas disease,parasitic eye infection, viral infection (e.g. HIV, herpes), bacterialinfection, Alzheimer's disease, hypertension, sepsis, acute leukemia,dystrophic epidermolysis bullosa, and muscular dystrophy.

[0004] In particular, protease/protease inhibitor imbalances are notablein a number of respiratory diseases. The classic and prototypic exampleof this is alpha 1-antitrypsin (AAT) deficiency, where low levels of AAT(also known as alpha 1 -protease inhibitor) in the bloodstream,determined by genetic factors, lead to decreased levels of AAT in thelung. The consequence of this is a decreased inhibitory capacity towardsthe proteolytic enzyme neutrophil elastase. This compromised ability tocontrol elastolytic activity, and the consequent degradation of lungelastin, leads inevitably to the early onset of pulmonary emphysema inmany individuals with AAT-deficiency.

[0005] Other respiratory diseases where protease/protease inhibitorimbalances have been shown to have significant involvement in diseaseprogression are asthma, chronic obstructive pulmonary disease (COPD),cystic fibrosis (CF), and respiratory distress syndrome. In asthma, themast cell-derived proteases, tryptase and chymase, have been shown to beinvolved in the inflammatory response to allergenic stimuli. Similarly,the inhibition of airway hyperresponsiveness by AAT has implicatedprotease targets for AAT, such as neutrophil elastase, cathepsin G, andkallikrein as possible contributory factors in this condition.

[0006] Elevated levels of several matrix metalloproteases (MMPs) havebeen shown in human COPD, particularly in COPD induced by cigarettesmoking. Although implicated in the degradation of lung collagen andelastin, the relative contributions of metalloproteases and elastase tothe reduction of lung elasticity, and consequent development ofpulmonary emphysema is not fully understood at the present time.Interestingly, transgenic mouse models for smoking-related emphysemahave implicated the murine metalloelastase (equivalent to human MMP-12)as a critical factor in the development of smoking related COPD in thisspecies.

[0007] Protease inhibitors are also implicated in the treatment of HIV.One of the major proteins coded for by HIV nucleic acid is a protease,and one of the most effective treatments of HIV to date is the use ofprotease inhibitors.

[0008] A full understanding of the equilibrium between the aboveproteases and their inhibitors is made even more complex by the findingsthat matrix metalloprotease inhibitors are capable of cleaving, andthereby inactivating AAT and, conversely, neutrophil elastase caninactivate endogenous tissue inhibitors of metalloproteases (TIMPs). Amajor pathogen of the lung, Pseudomonas aeruginosa, which colonizes thelungs of many individuals with CF, secretes a metalloelastase thatdegrades AAT, leading to much of the lung damage associated with CF.

[0009] The incidence of many diseases in which a protease/proteaseinhibitor imbalance is implicated is increasing; for example, theincidence of emphysema in the U.S. is up over 40% compared to 1982.While there have been advancements in the amelioration of thesediseases, there are at present no completely satisfactory treatments.Hence there is a need for improved methods of treatment to reducesymptoms and/or to slow or halt disease progress. The present inventionaddresses these needs.

SUMMARY OF THE INVENTION

[0010] This invention provides fusion proteins of protease inhibitors,or functionally active portions of protease inhibitors, such as fusionproteins comprising alpha 1-antitrypsin (AAT) and a second proteaseinhibitor. In some embodiments the invention provides fusion proteinscomprising AAT and secretory leukocyte protease inhibitor (SLPI), or AATand a tissue inhibitor of metalloproteases (TIMP), as well as methods ofmaking and using these fusion proteins. Such fusion proteins have theadvantage that they provide a broad spectrum of protease inhibition in asingle entity, which is useful in conditions where more than oneprotease is present, such as in many of the pathological conditionsdescribed above, and in tissue preparation, extraction, analysis, andother procedures where control of unwanted protease activity isdesirable.

[0011] In one aspect, the invention features a fusion protein comprisingAAT (including functionally active portions thereof) and anotherprotease inhibitor (including functionally active portions thereof),polynucleotides encoding the fusion protein, vectors and host cellscontaining the polynucleotides, methods of producing the fusion protein,and pharmaceutical compositions that contain the fusion protein. In oneaspect, the second protease inhibitor is a serine protease inhibitor, inanother aspect the second protease inhibitor is a tissue inhibitor ofmetalloproteases, in a further aspect the second protease inhibitor isan inhibitor of cysteinyl proteases, and in still a further embodiment,the second protease inhibitor is an inhibitor of aspartyl proteases.

[0012] In another aspect, the invention features a fusion proteincomprising AAT, or a functionally active portion thereof, fused to SLPI,or a functionally active portion thereof. Related to this aspect, theinvention includes polynucleotides encoding a fusion protein of AAT andSLPI, or functionally active portions thereof, vectors and host cellscontaining such polynucleotides, methods of producing such fusionproteins, and pharmaceutical compositions containing such fusionproteins.

[0013] In yet another aspect, the invention features a fusion proteincomprising AAT, or functionally active portion thereof, and TIMP, or afunctionally active portion thereof. An embodiment of this aspectincludes fusion proteins in which the TIMP portion is TIMP-1, or afunctionally active portion thereof. Related to this aspect, theinvention includes polynucleotides encoding such fusion proteins of AATand TIMP, or functionally active portions thereof, vectors and hostcells containing these polynucleotides, methods of producing the fusionproteins, and pharmaceutical compositions containing the fusionproteins.

[0014] In a further aspect, the invention features a fusion proteincomprising amino acids from about 1 to about 394 of AAT, and amino acidsfrom about 1 to about 107 of SLPI. In one embodiment of this aspect ofthe invention, the carboxy terminus of amino acids from about 1 to about394 of AAT is joined to the amino terminus of amino acids from about 1to about 107 of SLPI (i.e., the fusion protein comprises, from its aminoto its carboxy termini, amino acids from about 1 to about 394 of AATfused to amino acids from about 1 to about 107 of SLPI). In anotherembodiment the carboxy terminus of amino acids from about 1 to about 107of SLPI is joined to the amino terminus of amino acids from about 1 toabout 394 of AAT (i.e., the fusion protein comprises, from its amino toits carboxy termini, amino acids from about 1 to about 107 of SLPI fusedto amino acids from about 1 to about 394 of AAT). In a related aspect,the invention features the polynucleotides that encode the above fusionproteins. Also included are vectors and host cells containing thepolynucleotides that encode the fusion proteins, methods of producingthe fusion proteins, and pharmaceutical compositions that contain thefusion proteins.

[0015] In a further aspect, the invention features a fusion proteincomprising amino acids from about 1 to about 394 of alpha 1-antitrypsin;and amino acids from about 1 to about 184 of tissue inhibitor ofmetalloproteases-l. In one embodiment of this aspect of the invention,the carboxy terminus of amino acids from about 1 to about 394 of AAT isjoined to the amino terminus of amino acids from about 1 to about 184 oftissue inhibitor of metalloproteases-1 (i.e., the fusion proteincomprises, from its amino to its carboxy termini, amino acids from about1 to about 394 of AAT fused to amino acids from about 1 to about 184 oftissue inhibitor of metalloproteases- 1). In another embodiment thecarboxy terminus of amino acids from about 1 to about 184 of tissueinhibitor of metalloproteases-1 is joined to the amino terminus of aminoacids from about 1 to about 394 of AAT (i.e., the fusion proteincomprises, from its amino to its carboxy termini, amino acids from about1 to about 184 of tissue inhibitor of metalloproteases-1 fused to aminoacids from about 1 to about 394 of AAT). In a related aspect, theinvention features the polynucleotides that encode the above fusionproteins. Also included are vectors and host cells containing thepolynucleotides that encode the fusion proteins, methods of producingthe fusion proteins, and pharmaceutical compositions that contain thefusion proteins.

[0016] In yet a further aspect, the invention features a fusion proteincomprising amino acids from about 1 to about 394 of alpha 1-antitrypsin;and amino acids from about 1 to about 126 of tissue inhibitor ofmetalloproteases-1. In one embodiment of this aspect of the invention,the carboxy terminus of amino acids from about 1 to about 394 of AAT isjoined to the amino terminus of amino acids from about 1 to about 126 oftissue inhibitor of metalloproteases-1 (i.e., the fusion proteincomprises, from its amino to its carboxy termini, amino acids from about1 to about 394 of AAT fused to amino acids from about 1 to about 126 oftissue inhibitor of metalloproteases-1). In another embodiment thecarboxy terminus of amino acids from about 1 to about 126 of tissueinhibitor of metalloproteases-1 is joined to the amino terminus of aminoacids from about 1 to about 394 of AAT (i.e., the fusion proteincomprises, from its amino to its carboxy termini, amino acids from about1 to about 126 of tissue inhibitor of metalloproteases-1 fused to aminoacids from about 1 to about 394 of AAT). In a related aspect, theinvention features the polynucleotides that encode the above fusionproteins. Also included are vectors and host cells containing thepolynucleotides that encode the fusion proteins, methods of producingthe fusion proteins, and pharmaceutical compositions that contain thefusion proteins.

[0017] In still yet a further aspect, the invention features a fusionprotein comprising amino acids from about 1 to about 394 of alpha1-antitrypsin; and amino acids from about 1 to about 127 of tissueinhibitor of metalloproteases-1, wherein the alpha 1-antitrypsinpolypeptide is covalently linked to the tissue inhibitor ofmetalloproteases-1 polypeptide through a disulfide bond between aminoacid 127 of the tissue inhibitor of metalloproteases-1 polypeptide and afree cysteine residue of the alpha 1-antitrypsin polypeptide. In oneembodiment of this aspect of the invention, the free cysteine residue ofthe alpha 1-antitrypsin polypeptide is at position 232 in SEQ ID NO: 2.

[0018] Also within the invention are methods of using the proteaseinhibitor fusion proteins of the invention, including for inhibition ofprotease activity in vitro (e.g., in a biological sample) or in vivo(e.g., in treating diseases and conditions where the disease orcondition is associated with a protease/protease inhibitor imbalanceand/or an inflammatory response involving protease activity). Oneembodiment of this aspect of the invention includes a method forinhibiting protease activity by contacting the protease with one of thefusion proteins of the invention. In a further embodiment, the proteaseactivity is associated with a disorder selected from the groupconsisting of emphysema, asthma, chronic obstructive pulmonary disease,cystic fibrosis, otitis media, and otitis externa. In yet a furtherembodiment, the protease activity is associated with HIV infection. Inone embodiment, the fusion protein is contacted with the protease byadministering the fusion protein to an individual having the protease.

[0019] Another aspect of the methods of the invention is a method oftreating an individual suffering from, or at risk for, a disease ordisorder involving unwanted protease activity comprising administeringto the individual an effective amount of a fusion protein of theinvention. In one embodiment of this aspect, the individual suffers fromemphysema. In another embodiment of this aspect of the invention, theindividual suffers from asthma. In yet another embodiment of this aspectof the invention, the individual suffers from chronic obstructivepulmonary disease. In still yet another embodiment of this aspect of theinvention, the individual suffers from cystic fibrosis. In still yetfurther another embodiment of this aspect of the invention, theindividual suffers from otitis media or otitis externa.

[0020] Another aspect of the invention provides compositions and kitscomprising the fusion proteins of the invention. In one embodiment ofthis aspect of the invention, the kits further comprise instructions foruse of the fusion protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a schematic diagram of a yeast expression vector, pHG62,used to produce a SLPI/AAT fusion protein in the yeast Saccharomycescerevisiae. The resulting expressed protein is designated SLAPI.

[0022]FIG. 2 is a schematic diagram of a yeast expression vector, pHG62,used to produce a TIMP-1/AAT fusion protein in the yeast Saccharomycescerevisiae. The resulting expressed protein is designated TAPI.

[0023]FIG. 3 is a schematic diagram of a yeast expression vector, pKC65,used to produce a SLPI/AAT fusion protein (SLAPI) in the yeastSaccharomyces cerevisiae.

[0024]FIG. 4 is a schematic diagram of a yeast expression vector, pKC64,used to produce an AAT/TIMP-1 fusion protein in the yeast Saccharomycescerevisiae. The resulting expressed protease inhibitor is designatedreverse (r) TAPI.

[0025]FIG. 5 shows the inhibition of human neutrophil elastase (HNE) byrecombinant AAT, recombinant SLPI, and SLAPI.

[0026]FIG. 6 shows tryptase inhibition by SLPI, SLAPI, and AAT atvarious molar ratios.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates to fusion proteins of alpha 1-antitrypsin (AAT) and another protease inhibitor protein, and tomethods of making and using such fusion proteins to inhibit proteases.The second protease inhibitor of the fusion proteins of the inventionmay be an inhibitor of serine proteases, an inhibitor ofmetalloproteases, an inhibitor of cysteine proteases, or an inhibitor ofaspartate proteases. In one embodiment, the second protease inhibitor issecretory leukocyte protease inhibitor (SLPI). In another embodiment,the second protease inhibitor is a tissue inhibitor of metalloproteases(TIMP), preferably TIMP-1. The invention also includes thepolynucleotides encoding these constructs, the vectors comprising thepolynucleotides and host cells comprising the polynucleotides, as wellas methods of producing the fusion proteins. The invention also includesmethods of using the fusion proteins to inhibit proteases. The proteasesmay be present in vitro or they may be in an individual with apathology.

[0028] I. Definitions

[0029] As used herein, a “polynucleotide” is a polymeric form ofnucleotides of any length, which contain deoxyribonucleotides,ribonucleotides, and/or their analogs. The terms “polynucleotide” and“nucleotide” as used herein are used interchangeably. The term“polynucleotide” includes double-, single-stranded, and triple-helicalmolecules. Unless otherwise specified or required, any embodiment of theinvention described herein that is a polynucleotide encompasses both thedouble-stranded form and each of two complementary single-stranded formsknown or predicted to make up the double stranded form.

[0030] The following are non-limiting examples of polynucleotides: agene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs.Analogs of purines and pyrimidines are known in the art, and include,but are not limited to, aziridinylcytosine, 4-acetylcytosine,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, pseudoruacil, 5-pentynyluracil and 2,6-diaminopurine.The use of uracil as a substitute for thymine in a deoxyribonucleic acidis also considered an analogous form of pyrimidine.

[0031] If present, modification to the nucleotide structure may beimparted before or after assembly of the polymer. The sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsincluded in this definition are, for example, “caps”, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s).

[0032] Further, any of the hydroxyl groups ordinarily present in thesugars may be replaced by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH groups can be phosphorylatedor substituted with amines or organic capping groups moieties of from 1to 20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups.

[0033] Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, butnot limited to, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside.

[0034] As noted above, one or more phosphodiester linkages may bereplaced by alternative linking groups. These alternative linking groupsinclude, but are not limited to, embodiments wherein phosphate isreplaced by P(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”),P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing and ether (—O—) linkage, aryl, alkenyl, cycloalky,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical.

[0035] Although conventional sugars and bases will be used in applyingthe method of the invention, substitution of analogous forms of sugars,purines and pyrimidines can be advantageous in designing a finalproduct, as can alternative backbone structures like a polyamidebackbone.

[0036] The term “recombinant” polynucleotide (and by analogy, a“recombinant” polypeptide” produced by the expression of a recombinantpolynucleotide) is one which is not naturally occurring or is made bythe artificial combination of two otherwise separated segments ofsequence by chemical synthesis means or the artificial manipulation ofisolated segments of polynucleotides, e.g., by genetic engineeringtechniques. Thus, the term “recombinant” polynucleotide as used hereinintends a polynucleotide of genomic, cDNA, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation: (1) is notassociated with all or a portion of a polynucleotide with which it isassociated in nature, (2) is linked to a polynucleotide other than thatto which it is linked in nature, or (3) does not occur in nature.

[0037] The terms “polypeptide”, “oligopeptide”, “peptide” and “protein”are used interchangeably herein to refer to polymers of amino acids ofany length. The polymer may be linear or branched, it may comprisemodified amino acids, and it may be interrupted by non-amino acids. Theterms also encompass an amino acid polymer that has been modifiednaturally or by intervention; for example, disulfide bond formation,glycosylation, lipidation, acetylation, carboxylation, phosphorylation,ubiquitination, pegylation or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications. Such modifications are well known; see,e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, ed.Sambrook, et al., Cold Spring Harbor Laboratory Press (1989) or CurrentProtocols in Molecular Biology, ed. F. Ausubel et al., Greene Publishingand Wiley-Interscience: New York (1987 and periodic updates).

[0038] A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce thepolypeptide.

[0039] A “fusion protein” is a single polypeptide comprising regionsfrom two or more different proteins. The regions normally exist in or asseparate proteins and are brought together in the fusion protein. Theymay be linked together so that the amino acid sequence of one beginswhere the amino acid sequence of the other ends, or they may be linkedvia linker amino acids which are not normally a part of the constituentproteins. They may be linked in any manner, such as through amide bonds,disulfide bonds, etc. A fusion protein may contain more than one copy ofany of its constituent proteins or regions. The constituent proteins orregions may include the entire amino acid sequences of the proteins orportions of the amino acid sequences. As is apparent from the definitionof “protein,” above, the protein may be in branched form; e.g., the sidechain of one amino acid in one chain may be linked to the side chain ofanother, terminal amino acid in another chain by any of a variety ofmethods known to those of skill in the art (for example, disulfide bondformation). Alternatively, non-terminal amino acids of different chainsmay also be linked by intermolecular bonds between side chains (e.g.,disulfide bonds) to form a branched protein.

[0040] A “cell line” or “cell culture” denotes cells grown or maintainedin vitro. It is understood that the descendants of a cell may not becompletely identical (either morphologically, genotypically, orphenotypically) to the parent cell.

[0041] As used herein, the term “vector” refers to a polynucleotidemolecule capable of transporting another polynucleotide to which it hasbeen linked and can include a plasmid, cosmid or viral vector. The termincludes vectors that function primarily for insertion of apolynucleotide molecule into a cell, replication vectors that functionprimarily for the replication of polynucleotide, and expression vectorsthat function for transcription and/or translation of the DNA or RNA.Also included are vectors that provide more than one of the abovefunctions. The vector can be capable of autonomous replication or it canintegrate into a host DNA. Viral vectors include, e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses.

[0042] A vector can include nucleic acid coding for the fusion proteinof the invention in a form suitable for expression of the nucleic acidin a host cell. Preferably the recombinant expression vector includesone or more regulatory sequences operatively linked to the nucleic acidsequence to be expressed. The term “regulatory sequence” includespromoters, enhancers and other expression control elements (e.g.,polyadenylation signals). Regulatory sequences include those that directconstitutive expression of a nucleotide sequence, as well astissue-specific regulatory and/or inducible sequences. The design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or polypeptides, including fusionproteins or polypeptides, encoded by nucleic acids as described herein.

[0043] The recombinant expression vectors of the invention can bedesigned for expression of the fusion proteins of the invention inprokaryotic or eukaryotic cells. For example, polypeptides of theinvention can be expressed in E. coli, insect cells (e.g., usingbaculovirus expression vectors), yeast cells, mammalian cells inculture, or in transgenic animals. Suitable host cells are discussedfurther in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

[0044] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant protein.

[0045] A way to maximize recombinant protein expression in E. coli is toexpress the protein in host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

[0046] The fusion protein expression vector can also be a yeastexpression vector, examples of which are described herein, a vector forexpression in insect cells, e.g., a baculovirus expression vector or avector suitable for expression in mammalian cells in culture, or intransgenic animals. Methods of expressing proteins in yeast, such asSaccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, andKluyveromyces lactis, are well-known in the art.

[0047] When used in mammalian cells, the expression vector's controlfunctions are often provided by viral regulatory elements. For example,commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40.

[0048] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid). Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J, 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for example,the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

[0049] A “host cell” includes an individual cell or cell culture whichcan be or has been a recipient for vector(s) or for incorporation ofpolynucleotide molecules and/or proteins. A host cell can be anyprokaryotic or eukaryotic cell. For example, fusion proteins of theinvention can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art. Host cells include progeny of a single host cell,and the progeny may not necessarily be completely identical (inmorphology or in genomic of total DNA complement) to the original parentcell due to natural, accidental, or deliberate mutation. A host cellincludes cells transfected in vivo with a polynucleotide(s) of thisinvention.

[0050] Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation A “signal sequence,” also known as a “leader sequence,”is a short amino acid sequence that directs newly synthesized secretoryor membrane proteins to and through cellular membranes such as theendoplasmic reticulim. Signal sequences are typically in the N-terminalportion of a polypeptide and are cleaved after the polypeptide hascrossed the membrane.

[0051] As used herein, “treatment” is an approach for obtainingbeneficial or desired results, including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, alleviation of one or more symptoms, diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,preventing spread (i.e., metastasis) of disease, preventing occurrenceor recurrence of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared with expected survival if notreceiving treatment.

[0052] An “effective amount” is an amount sufficient to effectbeneficial or desired clinical results. An effective amount can beadministered in one or more administrations. In terms of treatment, an“effective amount” of a fusion protein of the invention is an amountthat is sufficient to palliate, ameliorate, stabilize, reverse or slowthe progression of a protease-associated disease state. An “effectiveamount” may be of a fusion protein used alone or in conjunction with oneor more agents used to treat a disease or disorder.

[0053] An “individual” is a vertebrate, preferably a mammal, morepreferably a human. Mammals include, but are not limited to, farmanimals, sport animals, primates, and pets.

[0054] “A,” “an,” and “the” include one or more.

[0055] “Comprising” means “including”

[0056] II. Fusion Proteins and their Construction

[0057] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology(including recombinant DNA techniques), microbiology, cell biology,biochemistry and immunology, which are within the skill of the art. Suchtechniques are explained fully in the literature, such as, “MolecularCloning: A Laboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Wei & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction”, (Mullis et al, eds., 1994); “Current Protocols in Immunology”(J. E. Coligan et al., eds., 1991).

[0058] These techniques are applicable to the production of thepolynucleotides and polypeptides of the invention, and, as such, are tobe considered when contemplating these inventive aspects. Particularlyuseful systems for individual aspects will be discussed below.

[0059] Fusion proteins of the invention are generally referred to byreference to the active components, e.g., a fusion protein of AAT andSLPI. It is understood that these references refer to variousembodiments, such as fusion proteins comprising functionally activeportions, etc.

[0060] Protein and nucleotides The compositions of the present inventioninclude fusion proteins and the polynucleotides which code for thesefusion proteins. The fusion proteins comprise AAT or a functionallyactive portion thereof and another protease inhibitor or a functionallyactive portion thereof.

[0061] A DNA sequence encoding human AAT (Table 1) and the amino acidsequence of human AAT (Table 2) are listed as SEQ ID NOS: 1 and 2,respectively. Functionally active portions of AAT and other proteaseinhibitors are known in the art and may be used in the fusion proteinsof the invention. Further, assays for assessing activity of functionallyactive portions (whether alone or in the context of a larger sequence)are known. Human AAT is the preferred form for the invention, and thenative amino acid sequence is the most preferred form. However,sequences from other species may be used. TABLE 1 DNA sequence encodinghuman AAT gaagaccctc aaggcgacgc cgctcaaaaa accgacacca 60 gtcatcacgaccaagaccat ccgactttta ataaaattac tccaaattta gccgaatttg 120 ctttttctttgtatagacaa ttagctcatc aaagtaattc tactaacatt ttttttagtc 180 ctgtttctattgccactgct ttcgccatgt tgagtttagg tactaaagcc gatacccatg 240 acgagattttagaaggttta aactttaatt tgaccgaaat cccagaagcc caaattcacg 300 agggttttcaagagttgttg agaactttga atcaacctga ttctcaattg caattaacta 360 ctggtaacggtttatttttg tctgaaggtt taaaattggt tgacaaattc ctagaagacg 420 tcaagaaactatatcatagt gaggctttta ccgttaattt tggtgatact gaggaagcta 480 aaaagcaaattaatgattat gttgagaaag gcacccaggg taagatcgtt gacctagtta 540 aagaattagatcgtgatacc gtcttcgcac tagttaacta tatttttttc aagggtaagt 600 gggaacgtcctttcgaggtt aaagatactg aagaggaaga ttttcatgtt gatcaagtta 660 ctactgtcaaagttccaatg atgaaaagac tgggtatgtt caatattcaa cattgcaaaa 720 aattaagttcttgggtctta ttaatgaagt atttaggtaa cgctactgct attttttttt 780 taccagacgaaggtaagctt caacatttag agaatgagtt gactcatgac attattacta 840 aatttttagagaacgaggat cgtcgtagcg cttctctgca cctgccaaag ttaagtatca 900 ccggtacttacgacttaaaa tctgttttag gccagttagg tattaccaaa gttttttcta 960 acggtgccgatttgagtggt gttactgaag aagctccatt aaaattgagt aaagctgttc 1020 acaaagccgtcttaactatt gatgaaaagg gtaccgaggc cgccggcgct atgttcctgg 1080 aagctattccaatgagcatt ccaccagaag ttaaatttaa taaaccattc gtttttctga 1140 tgatcgagcagaacactaaa agcccattgt ttatgggtaa ggttgtcaac ccaactcaga 1182 ag

[0062] TABLE 2 Amino acid sequence of human AAT Glu Asp Pro Gln Gly AspAla Ala Gln Lys Thr Asp  1               5                  10 Thr SerHis His Asp Gln Asp His Pro Thr Phe Asn         15                  20Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser 25                  30                  35 Leu Tyr Arg Gln Leu Ala HisGln Ser Asn Ser Thr             40                  45 Asn Ile Phe PheSer Pro Val Ser Ile Ala Thr Ala    50                  55                  60 Phe Ala Met Leu Ser LeuGly Thr Lys Ala Asp Thr                 65                  70 His AspGlu Ile Leu Glu Gly Leu Asn Phe Asn Leu         75                  80Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe85                  90                  95 Gln Glu Leu Leu Arg Thr LeuAsn Gln Pro Asp Ser             100                 105 Gln Leu Gln LeuThr Thr Gly Asn Gly Leu Phe Leu    110                 115                 120 Ser Glu Gly Leu Lys LeuVal Asp Lys Phe Leu Glu                   125                 130 AspVal Lys Lys Leu Tyr His Ser Glu Ala Phe Thr        135                 140 Val Asn Phe Gly Asp Thr Glu Glu Ala LysLys Gln 145                 150                 155 Ile Asn Asp Tyr ValGlu Lys Gly Thr Gln Gly Lys             160                 165 Ile ValAsp Leu Val Lys Glu Leu Asp Arg Asp Thr    170                 175                 180 Val Phe Ala Leu Val AsnTyr Ile Phe Phe Lys Gly                 185                 190 Lys TrpGlu Arg Pro Phe Glu Val Lys Asp Thr Glu         195                 200Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val205                 210                 215 Lys Val Pro Met Met Lys ArgLeu Gly Met Phe Asn             220                 225 Ile Gln His CysLys Lys Leu Ser Ser Trp Val Leu    230                 235                 240 Leu Met Lys Tyr Leu GlyAsn Ala Thr Ala Ile Phe                 245                 250 Phe LeuPro Asp Glu Gly Lys Leu Gln His Leu Glu         255                 260Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu265                 270                 275 Glu Asn Glu Asp Arg Arg SerAla Ser Leu His Leu             280                 285 Pro Lys Leu SerIle Thr Gly Thr Tyr Asp Leu Lys    290                 295                 300 Ser Val Leu Gly Gln LeuGly Ile Thr Lys Val Phe                 305                 310 Ser AsnGly Ala Asp Leu Ser Gly Val Thr Glu Glu         315                 320Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala325                 330                 335 Val Leu Thr Ile Asp Glu LysGly Thr Glu Ala Ala             340                 345 Gly Ala Met PheLeu Glu Ala Ile Pro Met Ser Ile350                 355                 360 Pro Pro Glu Val Lys Phe AsnLys Pro Phe Val Phe                 365                 370 Leu Met IleGlu Gln Asn Thr Lys Ser Pro Leu Phe         375                 380 MetGly Lys Val Val Asn Pro Thr Gln Lys 385                 390

[0063] Many protease inhibitors besides AAT have been described in theart, and any protease inhibitor (or functionally active portion) forwhich the amino acid sequence is known may be used as the partner to AATin the invention. (See, e.g., Aviles, F., ed, Innovations in proteasesand their inhibitors, W.deGruyter, Berlin, N.Y., 1993; Barrett, A. J.,and Salvesen, G, eds, Proteinase Inhibitors, Elsevier, Amsterdam, 1986;Bode, W., and Huber, R., Natural protein proteinase inhibitors and theirinteraction with proteinases, Eur. J. Biochem. 204:433-451, 1992; Bode,W., and Huber, R., Proteinase-protein inhibitor interactions,Fibrinolysis 8, Suppl. 1: 161-171, 1994, all of which are incorporatedherein by reference).

[0064] It will be readily understood by those of skill in the art thatthe native sequence is not necessarily required for a protein to befunctionally active. For example, a portion of the protein may be usedwhich retains the desired functionality; in the case of the proteins ofthe invention, this is generally a domain or domains of the proteinwhich are capable of inhibiting one or more proteases. Any such sequencemay be used, and any additional sequence may be provided, as long asthere is requisite functionality. The functionality need not be as highas the native protein, and thus in some instances may be reduced, thesame, or even enhanced as compared to the native protein, and it isunderstood that the functionality is generally assessed in the contextof the fusion protein.

[0065] In addition, it is well-understood in the art that amino acidchanges, including substitutions, deletions, insertions,post-translational modifications, and the use of amino acid analogs, maybe made in the native protein or a portion of the native protein withoutabolishing or significantly reducing the biological or immunologicalactivity of the protein. Single amino acids may be substituted forothers with the same charge or hydrophobicity. Other amino acids may besubstituted with amino acids of differing charge or hydrophobicitywithout significantly altering the function of the protein. It is alsocontemplated to use variants which enhance the function of the proteinas compared to native, or wild type, protein. In addition tosubstitutions, entire portions of the protein may be deleted withoutabolishing or significantly affecting the basic biological function ofthe protein, or extra amino acids inserted without abolishing orsignificantly affecting the function. Such changes are similar tochanges that occur by evolution, and the degree of similarity of twoproteins which differ in amino acid sequence can be determined by amethod of quantitative analysis such as that described by Pearson andLipman (Pearson, W. R., and Lipman, D. J., Proc. Natl. Acad. Sci. USA85:2444-2448, 1998), which compares the homology of amino acid sequencesas well as the substitutions of amino acids known to occur frequently inevolutionary families of proteins sharing a conserved function.

[0066] In the present invention, a “functionally active portion” of aprotease inhibitor is a protein that inhibits a protease and that has anamino acid sequence either identical to, or differing in at least oneamino acid from, the native form of the protein or a portion of thenative form. If the amino acid sequence is different from the nativeform, the functionally active portion nonetheless has greater similarityto the native sequence or a portion thereof, for example, as defined bythe above comparison algorithm of Pearson and Lipman, or other suchcomparison accepted in the art, than to the amino acid sequence of anyother natural polypeptide from the same species. For example, afunctionally active portion of AAT is a polypeptide which inhibitsneutrophil elastase, cathepsin G, and/or kallikrein, and which has anamino acid sequence which is either identical to the native AAT sequenceor a portion thereof or which is more similar to the native AAT sequenceor a portion thereof than it is to any other native human protein, forexample, as calculated by the algorithm of Pearson and Lipman. Suchfunctionally active portions of a native protein are often referred toas “analogs” of the protein (e.g., “SLPI analogs”), and the two termsare used synonymously herein.

[0067] A fusion protein that comprises a functionally active portion ofa protease inhibitor may contain additional sequences. For example,additions to the polypeptide chain at the C- or N-terminus may by usefulto facilitate purification by, for example, targeting the protein forextracellular secretion (see, for example, U.S. Pat. No. 4,870,008);such additions are generally cleaved after they have performed theirsignaling function, thus being a part of the DNA for the protein but nota part of the final protein. Such additions, as well as others, such asa sequence between the protease inhibitor polypeptides of the fusionprotein, can be included in the invention.

[0068] Each class of proteases has its own class of protease inhibitors.Thus, there are serine protease inhibitors, metalloprotease inhibitors,cysteine protease inhibitors, and aspartate protease inhibitors. Allknown naturally occurring protease inhibitors are proteins, except forsome secreted by microorganisms. This invention encompasses the proteinprotease inhibitors. As with the proteases themselves, the inhibitorscontain highly conserved regions and often have a great deal of homologyfrom member to member within a class.

[0069] The serine protease inhibitors include canonical inhibitors,non-canonical inhibitors, and serpins (see, for example, Otlewski, J.,Krowarsch, D., and Apostoluk, W., Protein inhibitors of serineproteases, Acta Biochim Polonica, 46:531-565, 1999). Canonicalinhibitors bind to the protease in the substrate binding site, and theirmechanism of inhibition resembles that of an ideal substrate.Non-canonical inhibitors contain an inhibitory N-terminus which binds tothe protease forming a parallel β-pleated sheet. Serpins, the majorprotease inhibitors in plasma, bind in a manner similar to canonicalinhibitors, but their mechanism of action involves the cleavage of asingle peptide bond. The serpins are a superfamily of inhibitors,consisting of a single chain with a conserved domain of 370-390 residues(see Potemka, J., Korzus, E, and Travis, J., The serpin superfamily ofproteinase inhibitors: structure function, and regulation, J. Biol.Chem. 269:15957-15960, 1994).

[0070] Both AAT and SLPI are serine protease inhibitors. AAT has beenstudied extensively, and the amino acid sequence of the protein wasreported by Carrell et al. (Nature 298: 329-334, 1982). The protein hasbeen produced by recombinant methods in yeast; see, e.g., Brake et al.,U.S. Pat. No. 4,752,576. The major physiological protease targets of AATinclude neutrophil elastase, cathepsin G, mast cell chymase, andkallikrein. Functionally active portions of AAT may also be used in thepresent invention, for example, those described in U.S. Pat. Nos.6,068,994 and 4,732,973, and in A. Hercz, Proteolytic cleavages inalpha-one antitrypsin and microheterogeneity, Biochem. Biophys. Res.Comm. 128: 199-203, 1985.

[0071] The DNA and amino acid sequences of human SLPI were reported byHeinzel et al. (Eur. J. Biochem. 160: 61-67, 1987), and are shown in SEQID NO: 3 and SEQ ID NO: 4, respectively (see Tables 3 and 4). Also,several patents describe SLPI, its nucleic acid, and/or functionallyactive portions of SLPI (see, e.g., U.S. Pat. Nos. 4,760,130; 5,464,822;4,845,076; 5,633,227; 5,851,983; 5,871,956; 5,900,400; 6,017,880; and6,291,662), any of which may be used in the invention. By using suchfunctionally active portions, one may adjust the inhibitory activity ofthe molecule to be more focussed on one or another of the proteases thenative molecule inhibits. Major protease targets of SLPI are neutrophilelastase, mast cell chymase and tryptase, and chymotypsin. TABLE 3 DNAsequence of human SLPI tctggaaagt ctttcaaggc cggtgtttgt ccaccaaaga 60agtccgctca atgtttgaga tacaagaagc cagaatgtca atccgactgg caatgtccag 120gtaagaagag atgttgtcca gacacttgtg gtatcaagtg tctagaccca gttgacaccc 180caaacccaac tagaagaaag ccaggtaagt gtccagttac ttacggtcaa tgtttgatgt 240tgaacccacc aaacttctgt gaaatggacg gtcaatgtaa gagagacttg aagtgttgta 300tgggtatgtg tggtaagtcc tgtgtttccc cagtcaaggc c 321

[0072] TABLE 4 Amino acid sequence of human SLPI Ser Gly Lys Ser Phe LysAla Gly Val Cys Pro Pro  1               5                  10 Lys LysSer Ala Gln Cys Leu Arg Tyr Lys Lys Pro         15                 20Glu Cys Gln Ser Asp Trp Gln Cys Pro Gly Lys Lys25                  30                  35 Arg Cys Cys Pro Asp Thr CysGly Ile Lys Cys Leu             40                  45 Asp Pro Val AspThr Pro Asn Pro Thr Arg Arg Lys    50                  55                  60 Pro Gly Lys Cys Pro ValThr Tyr Gly Gln Cys Leu                 65                  70 Met LeuAsn Pro Pro Asn Phe Cys Glu Met Asp Gly         75                  80Gln Cys Lys Arg Asp Leu Lys Cys Cys Met Gly Met85                  90                  95 Cys Gly Lys Ser Cys Val SerPro Val Lys Ala            100                 105

[0073] In the fusion proteins of the present invention, theAAT-containing sequence may be joined C-terminally to the N-terminus ofthe second protease inhibitor (or functionally active portion), such asSLPI, or SLPI may be fused C-terminally to the N-terminus of AAT. Thefusion of the two proteins of the fusion protein may be by means of asimple peptide bond, or there may be one or more additional amino acidswhich comprise the fusion linkage between the two proteins of the fusionprotein. In a preferred embodiment, there is a methionine between theAAT and the SLPI. There may be additional sequence(s) in one or morelocations of the fusion proteins of the invention. Further, it isunderstood that the relative orientation of each protease inhibitorcomponent (e.g., AAT and second protease inhibitor) encompasses generalorientation with respect to C terminus/N terminus, and this encompassesdirect linkage of components as well as additional sequence(s) linkingcomponents.

[0074] In one embodiment of the invention, AAT or a functionally activeportion thereof is linked to a metalloprotease inhibitor, or afunctionally active portion thereof. Of the metalloproteases, the matrixmetalloproteases (MMPs) have been found to be particularly important ina number of normal and pathological conditions. The MMPs, which comprisethe collagenases, gelatinases, and stromelysin, have similar structures,with a propeptide, an amino terminal domain, a fibronectin-like domain,a zinc-binding domain, , and a C-terminal domain. In addition, somemembers incorporate a transmembrane domain and a α2V collagen-likedomain. The MMPs are inhibited by the tissue inhibitors of matrixmetalloproteases, or TIMPs, which are present in all connective tissue.There are four known human TIMPs, referred to as TIMP-1, TIMP-2, TIMP-3,and TIMP-4, which share sequence homology to a consensus sequence. Allof these TIMPs (including functionally active fragments, variants, etc.)are encompassed within the invention. TIMP-1 is 43% homologous to theconsensus sequence, TIMP-2 is 62% homologous, TIMP-3 is 56% homologous,and TIMP-4 is 61% homologous. The amino acid and nucleotide sequences ofall four human TIMPs have been characterized: TIMP-1 (Docherty et al.,Nature 318: 66-69, 1985), the DNA and amino acid sequences of which areshown in SEQ ID NOs: 5 and 6, respectively (see Tables 5 and 6); TIMP-2(Boone et al., Proc. Natl. Acad. Sci. 87:2800-2804), TIMP-3 (Wilde etal., DNA Cell Biol. 13: 711-718); TIMP-4 (Hawkins et al., U.S. Pat. No.5,643,752). The TIMPs are considered a single class based on their aminoacid sequence homology, the fact that each contains 12 cysteines and sixdisulfide bonds, their ability to inhibit metalloproteases, and thepresence of the VIRAK motif which interacts with the metal ion in ametalloprotease. There are both differences and overlap in the proteaseinhibitory activities of the TIMPs. TIMP-1 inhibits activatedinterstitial collagenase, the 92 kDa Type IV collagenase, andstromelysin, TIMP-2 inhibits gelatinases A and B as well as the 72 kDAType IV collagenase, TIMP-3 inhibits collagenase 1, stromelysin, andgelatinases A and B. TIMP-4 appears to inhibit gelatinase andcollagenase. TABLE 5 DNA sequence of human TEMP-1 tgcacctgtg tcccaccccacccacagacg gccttctgca 60 attccgacct cgtcatcagg gccaagttcg tggggacaccagaagtcaac cagaccacct 120 tataccagag ttatgagatc aagatgacca agatgtataaagggttccaa gccttagggg 180 atgccgctga catccggttc gtctacaccc ccgccatggagagtgtctgc ggatacttcc 240 acaggtccca caaccgcagc gaggagtttc tcattgctggaaaactgcag gatggactct 300 tgcacatcac tacctgcagt ttcgtggctc cctggaacagcctgagctta gctcagcgcc 360 ggggcttcac caagacctac actgttggct gtgaggaatgcacagtgttt ccctgtttat 420 ccatcccctg caaactgcag agtggcactc attgcttgtggacggaccag ctcctccaag 480 gctctgaaaa gggcttccag tcccgtcacc ttgcctgcctgcctcgggag ccagggctgt 540 gcacctggca gtccctgcgg tcccagatag cc 552

[0075] TABLE 6 Amino acid sequence of human TIMP-1 Cys Thr Cys Val ProPro His Pro Gln Thr Ala Phe  1               5                  10 CysAsn Ser Asp Leu Val Ile Arg Ala Lys Phe Val        15               20Gly Thr Pro Glu Val Asn Gln Thr Thr Leu Tyr Gln25                  30                  35 Arg Tyr Glu Ile Lys Met ThrLys Met Tyr Lys Gly             40                  45 Phe Gln Ala LeuGly Asp Ala Ala Asp Ile Arg Phe    50                  55                  60 Val Tyr Thr Pro Ala MetGlu Ser Val Cys Gly Tyr                 65                  70 Phe HisArg Ser His Asn Arg Ser Glu Glu Phe Leu         75                  80Ile Ala Gly Lys Leu Gln Asp Gly Leu Leu His Ile85                  90                  95 Thr Thr Cys Ser Phe Val AlaPro Trp Asn Ser Leu             100                 105 Ser Leu Ala GlnArg Arg Gly Phe Thr Lys Thr Tyr    110                 115                 120 Thr Val Gly Cys Glu GluCys Thr Val Phe Pro Cys                 125                 130 Leu SerIle Pro Cys Lys Leu Gln Ser Gly Thr His         135                 140Cys Leu Trp Thr Asp Gln Leu Leu Gln Gly Ser Glu145                 150                 155 Lys Gly Phe Gln Ser Arg HisLeu Ala Cys Leu Pro             160                165 Arg Glu Pro GlyLeu Cys Thr Trp Gln Ser Leu Arg    170                 175             180 Ser Gln Ile Ala

[0076] The complete structure of a TIMP is not necessarily a requirementfor metalloprotease inhibition. For example, functionally activeportions of TIMP-2 (often referred to as “TIMP-2 analogs”) have beenprepared that retain their inhibitory activity toward metalloproteases(see Willenbrock et al., Biochemisty 32: 4330-4337, 1993). A partialsequence of TIMP-1 that contains only the first three loops of themolecule is capable of inhibiting matrix metalloproteases. (Note: thewords “metalloprotease” and “metalloproteinase” are synonymous and bothare used when referring to these enzymes in the literature; forconsistency we will use only “metalloprotease” herein). The N-terminusof the TIMP molecule is where the inhibitory activity is found, and theinhibitory mechanisms appear to involve several specific amino acidsequences. (see, for example, Murphy, G. Houbrechts, A., Cockett, M. I.,Williamson, R. A., O'Shea, M., and Docherty, AJP, The N-terminal domainof TIMP retains metalloprotease activity. Biochemistry 30: 8097-8102,1991; Woessner, J., Matrix metalloproteases and their inhibitors inconnective tissue remodeling. FASEB J5: 2145-2154, 1991, and EPApublication # 0648838 A1, Tissue inhibitor of metalloprotease type three(TIMP-3), Silbiger and Koski). One preferred N-terminal fragment ofTIMP-1 for construction of some embodiments of the present invention isthe first 126 N-terminal amino acids of the native form. In makingconstructs this fragment is often used with an initial methionine, andthus contains 127 amino acids (the initial methionine plus theN-terminal 1-126 amino acids of TIMP-1); this fragment is referred to asN-TIMP 1-127 (see SEQ ID NO: 22 and Table 30). Another preferredN-terminal fragment of TIMP-1 for construction of other embodiments ofthe present invention is the first 127 N-terminal amino acids of thenative form. Amino acid 127 of this fragment is a free cysteine, and isthus available to participate in disulfide bond formation, which is onemanner of constructing the fusion proteins of the invention. In makingconstructs this fragment is often used with an initial methionine, andthus contains 128 amino acids (the initial methionine plus theN-terminal 1-127 amino acids of TIMP-1); this fragments is referred toas N-TIMP 1-128 (see SEQ ID NO: 24 and Table 32).

[0077] In the AAT-TIMP fusion proteins of the present invention, AAT maybe linked C-terminally to the N-terminus of TIMP, or TIMP may be fusedC-terminally to the N-terminus of AAT. The fusion of the two proteins ofthe fusion protein may be by means of a simple peptide bond, or theremay be one or more additional amino acids which comprise the fusionlinkage between the two proteins of the fusion protein.

[0078] Cysteine proteases are inhibited by the cystatins, stefins, andkininogens. The cystatins and stefins consist of an α-helix surroundedby a five-stranded antiparallel β-pleated sheet, forming a wedge that iscomplementary to the active site of the protease. At one end of theβ-pleated sheet is a highly conserved β-hairpin loop with the sequenceQVVAG (SEQ ID. NO: 11)(see Barrett et al., in Proteinase inhibitors(Barret, A. J. and Salvesen, G., eds) pp. 515-569, Elsevier, Amsterdam,1986, and Turk, V., and Bode, W., FEBS Lett. 285:213-219, 1991. Oneembodiment of the present invention is AAT or a functionally activeportion thereof linked to a cystatin, stefin, or kininogen orfunctionally active portion thereof.

[0079] Aspartyl proteases include the HIV aspartyl protease, renin(involved in hypertension), pepsin, cathepsin D (implicated in tumormetastasis), and aspartyl hemoglobinases (from the malarial parasite).

[0080] The HIV protease cleaves polyprotein precursors to the functionalproteins of the virion core in the final stages of viral maturation. Itsinhibition has been a major target for HIV and AIDS treatment (Huof, J.R., “HIV protease: a novel chemotherapeutic target for AIDS,” J. Med.Chem. 34:2305-2314). Several HIV protease inhibitors, includingritonavir, Crixivan, and saquinavir, have been approved by the FDA forHIV treatment. Cathepsin D is a lysosomal protein which normally isinvolved in the degradation of intracellular or phagocytosed proteins.However, it has been implicated in a number of diseases. For example,cathepsin D may degrade the extracellular matrix and promote the escapeof cancer cells in metastasis and invasion of new tissues, and alsoappears to be an agent in pathological brain changes such as those seenin Alzheimer's disease. Elevated levels of cathepsin D have beenobserved in cerebrospinal fluid of Alzheimer's patients, and it isassociated with the cleavage of the amyloid-β-protein precursor. Themalarial parasite aspartyl proteases, Plasmepsins I and II, are highlyhomologous with human cathepsin D, and are essential in the breakdown ofhemoglobin to products which the parasite uses for nutrition. Inhibitorsof these proteases kill the parasite in cell culture of infected humanerythrocytes. Renin is an enzyme originating in the kidney whichconverts angiotensinogen to angiotensin, a crucial event in therenin-angiotensin modulation of blood pressure, ultimately resulting inthe production of angiotensin II which is a powerful vasoconstrictor.Hence, inhibitors of renin have long been considered as candidates forthe control of hypertension.

[0081] The most well-known of the natural aspartyl protease inhibitorsis pepstatin, a peptide originally isolated from a culture ofstreptomyces, with the formulaisovaleryl-L-valyl-L-valyl-statyl-L-alanyl-statin, (SEQ ID NO: 12) inwhich “statin” is the unusual amino acid(3S,4S)-4-amino-3-hydroxy-6-methyl-heptanoic acid. Pepstatin is activeagainst pepsin, cathepsin D, and renin. In addition to pepstatin, manyprotease inhibitors targeted at the various aspartyl proteases have beendesigned and produced, often based on the structure of pepstatin (seeU.S. Pat. No. 4,746,648; Umezawa, H, et al., Pepstatin, a new pepsininhibitor produced by Actinomycetes. J Antibiot (Tokyo) 23:259-62, 1970;Morishima, H., et al., The structure of pepstatin. J Antibiot (Tokyo)23:263-5, 1970; Lin, Ty and Williams, H R., Inhibition of cathepsin D bysynthetic oligopeptides. J. Biol. Chem. 254:11875-83, 1979; Jupp, R A,et al., The selectivity of statin-based inhibitors against various humanaspartic proteinases, Biochem. J. 265:871 -8, 1990; Agarwal, N S andRich, D H, Inhibition of cathepsin D by substrate analogues containingstatine and by analogues of pepstatin, J. Med. Chem. 29:2519-24, 1986;Baldwin, E T, et al., Crystal structures of native and inhibited formsof human cathepsin D: implications for lysosomal targeting and drugdesign. Proc. Natl. Acad. Sci., USA 90: 6796-800, 1993; Francis, S E etal., Molecular characterization and inhibition of a Plasmodiumfalciparum aspartyl hemoglobinase, EMBO J 13: 306-17, 1994). Oneembodiment of the present invention is AAT or a functionally activeportion thereof linked to an aspartyl protease inhibitor, such aspepstatin, or a functionally active portion thereof.

[0082] Production of the fusion proteins, polynucleotides, and hostcells of the invention. Ordinarily, production of the fusion proteins ofthe present invention is accomplished by constructing the appropriatepolynucleotide (generally DNA) sequence and expressing it in recombinantcell culture. Alternatively, however the polypeptides of this inventionmay be synthesized according to other known methods. Techniques forsynthesis of polypeptides are described, for example, in Merrifield, J.Amer. Chem. Soc. 85:2149-2156, 1963. Polypeptide chains containingprotease-inhibiting domains may be joined by a peptide bond, or by linksbetween amino acid side chains, e.g., disulfide bonds, by methodswell-known to those of skill in the art.

[0083] The recombinant fusion proteins are produced by an expressionvector or plasmid comprising DNA segments that direct the synthesis ofthe fusion protein, also in accordance with the present invention. Suchpolynucleotides include RNA, cDNA, genomic DNA, synthetic forms, andmixed polymers, both sense and antisense strands. Such polynucleotidescan be chemically or biochemically modified and can contain non-naturalor derivatized nucleotide bases. The sequence encoding the fusionpolypeptide can be interrupted by introns. The polynucleotide sequencesof this invention are of a length sufficient to encode such a fusionpolypeptide and, if necessary, any vector sequences. The sequences areusually several hundred nucleotides or nucleotide base pairs in lengthand may be several kilobases long. In other embodiments in which joiningof protease inhibitors is via one or more disulfide bonds, thepolynucleotides encoding the polypeptide chains of the individualinhibitors may be expressed separately, generally by being in separatevectors.

[0084] The present invention also encompasses methods for producing thefusion proteins, and pharmaceutical compositions containing the fusionproteins.

[0085] Techniques for polynucleotide manipulation, including theconstruction of polynucleotides capable of encoding and expressing thefusion polypeptides of the present invention, are well known and aredescribed generally, for example, in Sambrook et al., op. cit., orAusubel et al., op. cit. Reagents useful in applying such techniques,such as restriction enzymes and the like, are widely known in the artand commercially available.

[0086] The recombinant polynucleotide sequences used to produce fusionpolypeptides of the present invention (or used to produce antisensesequences) may be derived from natural or synthetic sequences. Toconstruct the fusion proteins by recombinant methods, the appropriatepolynucleotide sequences are operably linked. A polynucleotide sequenceis “operably linked” when it is in a functional relationship withanother polynucleotide sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects its transcription orexpression. Generally, operably linked means that the DNA sequencesbeing linked are contiguous and, where necessary to join two polypeptidecoding regions, contiguous and in reading frame. Sequences coding forsignal peptides (i.e., leader sequences) may also be added to therecombinant polynucleotide sequences, so that the polypeptide chainproduced is routed to the appropriate intracellular or extracellularspace for further manipulation, e.g., extraction and purification. Thesignal peptides from, for example, AAT (SEQ ID NOS:25 and 26, see Tables7 and 8, for DNA and amino acid sequences, respectively ), SLPI (SEQ IDNOS:27 and 28, see Tables 9 and 10, for DNA and amino acid sequences,respectively) TIMP-1 (SEQ ID NOS:29 and 30, see Tables 11 and 12, forDNA and amino acid sequences, respectively), α-factor signal (yeast)(SEQ ID NOS:31 and 32, see Tables 13 and 14, for DNA and amino acidsequences, respectively) , human serum albumin signal, or other proteinscan be used to signal the secretion of these proteins from various celllines, using methods known in the art. TABLE 7 DNA for leader sequencefor human AAT (Kurachi, K. et al, 1981, Proc Natl. Acad. Sci 78,p.6826.) ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTG 60CTGCCTGGTCCCT GTCTCCCTGGCT 73

[0087] TABLE 8 Amino acid sequence of leader sequence for human AAT(Kurachi, K. et al, 1981, Proc Natl. Acad. Sci 78, p.6826.)MPSSVSWGILLLAGLCCLVPVSLA 24

[0088] TABLE 9 DNA for leader sequence for human SLPI (Heinzel, R. etal., 1986, Eur. J. Biochem. 160,p. 61.)ATGAAGTCCAGCGGCCTCTTCCCCTTCCTGGTGCTGCTTGCCCTGGG 60 AACTCTGGCACCTTGGGCTGTGGAAGGC 75

[0089] TABLE 10 Amino acid sequence of leader sequence for human SLPI(Heinzel, R. et al., 1986, Eur. J. Biochem. 160,p. 61.)MKSSGLFPFLVLLALGTLAPWAVEG 25

[0090] TABLE 11 DNA for leader sequence for human TIMP-1 (Do- cherty, AJet al, 1985, Nature 318, p. 66) -ATGGCCCCCTTTGAGCCCCTGGCTTCTGGCATCCTGTTGTTGCTGTG 60 GCTGATAGCCCCCAGCAGGGCC 70

[0091] TABLE 12 Amino acid sequende of leader sequence for human TIMP-1(Docherty, AJ et al, 1985, Nature 318, p. 66) MAPFEPLASGILLLLWLIAPSRA 23

[0092] TABLE 13 DNA fof leader sequence for alpha factor from S.cervisiae (Kurjan,J. and Herskowitz,I., 1982, Cell 30, p. 933)ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTC 60 CGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGA 120 AGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTC 180 CAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAA 240 AGAAGAAGGGGTATCTCTAGATAAAAGAGAGGCTGAAGCTTG 269

[0093] TABLE 14 Amino acid sequence of leader sequence for alpha factorfrom S.cervisiae (Kurjan, J. and Herskowitz,I., 1982, Cell 30, p. 933)MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGD 60 FDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEA 89

[0094] The polynucleotide sequences given for the constituent proteinsof the fusion proteins of the present invention represent only oneexample of the polynucleotide sequences that may be used in the presentinvention. Because the genetic code is degenerate, more than one codonmay be used to code for a given amino acid, and there will be manydifferent DNA sequences which code for the same polypeptide sequence.The use of non-naturally-occurring codons in the nucleotide sequencescoding for the desired fusion proteins may be advantageous in thatdifferent codons, which may be preferred by different prokaryotic oreukaryotic hosts (see Murray, E. E., Nuc. Acids Res. 17:477-508, 1989),may be used to modify the expression of the fusion protein in a varietyof desirable ways. These include increasing the rate of expression ofthe fusion protein, or producing RNA transcripts having a longerhalf-life than those produced from DNA containing thenaturally-occurring codons.

[0095] The polynucleotides of the present invention are optionallyproduced by chemical synthesis, e.g., by the phosphoramidite methoddescribed by Beaucage and Carruthers (Tetra, Letts. 22:1859-1862, 1981)or the triester method according to Matteucci et al. (J. Am. Chem. Soc.103:3185, 1981). Chemical synthesis may be performed on commercialautomated oligonucleotide synthesizers.

[0096] Large amounts of the polynucleotides of the present invention maybe produced by replication in a suitable host cell, whether bacterial,yeast, such as Saccaromyces cerevisiae, insect, amphibian, avian,mammalian or other cells and expression systems. The natural orsynthetic polynucleotide (such as DNA) fragments coding for a desiredfragment will be incorporated into recombinant polynucleotideconstructs, typically DNA constructs.

[0097] These constructs are introduced into prokaryotic or eukaryoticcells where they replicate. Usually the constructs are suitable forautonomous replication in a unicellular host, such as yeast or bacteria.A preferred host cell for the present invention is yeast, for exampleSaccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, andKluyveromyces lactis. The constructs also can be introduced to andintegrated within the genome of a cultured insect, mammalian, plant orother eukaryotic cell lines. Suitable methods for these purposes arewell known in the art and have been described, e.g., in Sambrook et al.(1989) or Ausubel et al. (1987 and periodic updates).

[0098] Polynucleotide constructs prepared for introduction into aprokaryotic or eukaryotic host typically comprise a replication systemrecognized by the host, including the intended DNA fragment encoding thedesired polypeptide, and preferably also include transcription andtranslational initiation regulatory sequences operably linked to thepolypeptide encoding segment. Expression vectors include, for example,an origin of replication or autonomously replicating sequence (ARS) andexpression control sequences, a promoter, an enhancer and necessaryprocessing information sites, such as ribosome-binding sites, RNA splicesites, polyadenylation sites, transcriptional terminator sequences, andmRNA stabilizing sequences. Such vectors are prepared by means ofstandard recombinant techniques well known in the art and discussed, forexample, in Sambrook et al. (1989) or Ausubel et al. (1987).

[0099] Appropriate promoter and other necessary vector sequences areselected to function in the host. Examples of functional combinations ofcell lines and expression vectors are described in Sambrook et al., 1989or Ausubel et al., 1987); see also, e.g., Metzger et al., Nature334:31-36, 1988. Many useful vectors are known in the art and arecommercially available. For use in prokaryotic hosts, promoters includebut are not limited to the trp, lac and phage promoters, tRNA promotersand glycolytic enzyme promoters. Useful yeast promoters include but arenot limited to the promoter regions for metallothionein,3-phosphoglycerate kinase or other glycolytic enzymes such as enolase orglyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. A preferred promoter and terminatorin yeast is ADH2. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman et al. EP 73,657A.Appropriate nonnative mammalian promoters include but are not limited tothe early and late promoters from SV40 (Fiers et al. Nature 273:113,1978) or promoters derived from murine molony leukemia virus, mousemammary tumor virus, avian sarcoma viruses, adenovirus II, bovinepapilloma virus and polyoma virus. In addition, the construct can bejoined to an amplifiable gene (e.g., DHFR) so that multiple copies ofthe gene are made.

[0100] Such expression vectors can replicate autonomously.Alternatively, the expression vector can replicate by being insertedinto the genome of the host cell, by methods well known in the art.

[0101] Expression and cloning vectors generally include a selectablemarker, which encodes a polypeptide necessary for the survival or growthof its host cells. This gene's presence ensures the growth of only hostcells expressing the marker. Typical selection genes encode polypeptidesthat (a) confer resistance to antibiotics or other toxic substances,e.g. ampicillin, neomycin, methotrexate, etc.; (b) complementauxotrophic deficiencies; or (c) supply critical nutrients not availablefrom complex media. The choice of the proper selectable marker dependson the host cell. A preferred marker in yeast host cells is the URA3gene, which provides a selectable marker in the yeast 2 micron plasmidfor autonomous replication in yeast. Appropriate markers for differenthosts are well known in the art.

[0102] One of skill in the art will recognize that there are a number oftypes of host cell, both prokaryotic and eukaryotic, that will besuitable for expression of the fusion proteins of the present invention.The most commonly used prokaryotic hosts are strains of E. coli,although other prokaryotes, such as Bacillus subtilis or Pseudomonas,may also be used. Mammalian or other eukaryotic host cells, such asthose of yeast, filamentous fungi, plant, insect, amphibian or avianspecies, may also be useful for production of the polypeptides of thepresent invention, as well as the COS, CHO and HeLa cells lines andmyeloma cell lines. The preferred host cell type for the presentinvention is yeast, such as Saccharomyces cerevisiae, Pichia pastoris,Hansenula polymorpha, and Kluyveromyces lactis. Each type of host cellsrequires that the recombinant protein gene be operably linked toappropriate expression control sequence. Such control sequences includea promoter such as the T7, trp, or lambda promoters, a ribosome bindingsite and preferably a transcription termination signal, for E. coli, ora promoter and preferably an enhancer derived from immunoglobulin genes,SV40, cytomegalovirus, etc., and a polyadenylation sequence, foreukaryotic cells, and may include splice donor and acceptor sequences.

[0103] Vectors with the polynucleotides of interest can be transcribedin vitro, and the resulting RNA are introduced into host cells by wellknown methods (e.g. by injection). See, T. Kubo et al., FEBS Lett.241:119, 1988. Alternately, the vectors can be introduced directly intohost cells by methods well known in the art, which vary depending on thetype of cellular host. These methods include but are not limited toelectroporation; transfection employing lithium acetate, which is thepreferred method, or calcium chloride, rubidium chloride calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent, such as a retroviral genome). See generally, Sambrooket al. (1989) and Ausubel et al. (1987). The so-transformed cells arealso meant to include the progeny of such cells.

[0104] The invention as claimed does not call for a purified protein,and purification need be carried out only to the level of purityappropriate for the desired use of the proteins. Standard purificationtechniques, well-known in the art, may be used to purify the proteinsafter expression, including affinity columns, ammonium sulfateprecipitation, column chromatography, gel electrophoresis and the like.Such techniques are described in, for example, R. Scopes, “ProteinPurification”, Springer-Verlag, N.Y. (1982).

[0105] Assay of the fusion proteins. The activities of the proteaseinhibitors may be assessed by means known in the art for each of theindividual protease inhibitors; in general, one assays the activity ofthe appropriate protease in the presence and in the absence of theinhibitor. See, e.g., Barrett, Alan J., ed. Proteolytic enzymes: serineand cysteine peptidases. Meth Enz Vol. 244, San Diego, Academic Press,1994.

[0106] Preferred assay methods determine AAT activity by inhibition ofporcine pancreatic elastase in a microtiter plate format, or byinhibition of human neutrophil elastase, SLPI tryptase-inhibitingactivity by HPLC-based methods, and matrix metalloprotease activities,as described in Examples.

[0107] For SLPI, an assay is commonly based on the ability of thesubstance to inhibit a serine protease, preferably leukocyte orpancreatic elastase. The inhibitor is mixed with a known concentrationof protease and the residual enzyme activity is assessed by its abilityto hydrolyze methoxysuccinyl-alanyl-alanyl-prolyl-valine-p-nitroanilide,as described by T. Teshima et al., J. Biol. Chem. 257: 5085-91, 1982,and K. Nakajima et al., J. Biol. Chem 254:4027-32, 1979. Thedissociation constant, Ki, of the complex formed from the interaction ofthe inhibitor with leukocyte or porcine pancreatic elastase may beobtained by standard kinetic methods. In addition, AAT, SLPI and thefusion proteins of the present invention may be assayed for theirabilities to inhibit airway hyperresponsiveness in an animal model (e.g.allergen-challenged sheep).

[0108] Assays for the TIMPs have been described in U.S. Pat. Nos.5,595,885 and 5,643,752 and EP Nos. 0404750B1 and 0648838A1. Theseassays involve the inhibition of a collagenase or a gelatinase by theTIMP, usually by assessing the collagenase or gelatinase activity by itsability to digest a gelatin, which can be, for example, ¹⁴C labeled (seeCollier et al, J. Biol. Chem., 263: 6579-81,1988; and Wilhelm et al.,Proc. Natl. Acad. Sci. USA 84: 6725-6729, 1987). In addition, AAT, theTIMPs, and the fusion proteins of the present invention may be assayedfor the inhibition of development of emphysema in murine models. Suchassays may include standard laboratory strains of mice, ortransgenically modified mice that are treated with cigarette smoke overextended periods.

[0109] Aspartyl protease inhibitors are assayed according to theprotease of interest. HIV-1 protease inhibition may be assayed by themethod described essentially by M. W. Pennington et al., Peptides 1990,Gimet, E. and Andrew, D., eds, Escom; Leiden, Netherlands, (1990).Pepstatin-like inhibitors may be assayed by the method of Guyene, T T,et al., Inhibition of human plasma renin activity by pepstatin, J. Clin.Endocrinol. Metab., 43:1301-6, 1976, described in U.S. Pat. No.4,746,648. For inhibitors of cathepsin D and plasmepsins, the assaymethod described in U.S. Pat. No. 5,849,691 may be used.

[0110] III. Pharmaceutical Compositions

[0111] The proteins of the invention, fragments thereof, as well nucleicacids of the invention (also referred to herein as “active compounds”)can be incorporated into pharmaceutical compositions. Such compositionstypically include the protein or nucleic acid and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Supplementary active compounds can also be incorporated into thecompositions.

[0112] A pharmaceutical composition is formulated to be compatible withits intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral, inhalation, transdermal (topical), transmucosal, andrectal administration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0113] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0114] Sterile injectable solutions can be prepared by incorporating theactive compound in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0115] Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0116] The compounds of the invention also can be formulated into aslow-release (e.g., sustained delivery) formulation, for example aformulation that allows release of the compound over days, weeks ormonths, using slow-release formulations known in the art for delivery ofproteinaceous compounds are known in the art. Furthermore, the compoundsof the invention can be formulated to protect them from proteasedegradation.

[0117] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide.Alternatively, the compounds are delivered using a CFC-free metered doseinhaler, or a nebulizer. Dry proteins in carriers such as mannitol,sucrose, or lactose may be delivered in a spray to the lower airwayepithelia, which are permeable to proteins up to about MW 20 kDa.Particles of approximately one micron in diameter may be delivered tothe distal alveolar surface via dry powder inhalers, such as thosedesigned by Inhale, Dura, and other manufacturers known to those ofskill in the art. Ultrasonic nebulizers may be used to deliversolutions, with or without liposomes. Large porous particles are also aneffective method for pulmonary delivery using dry powder. Furtherdiscussion of pulmonary delivery of drugs maybe found in McElvaney, etal., J. Clin Invest. 90:1296-1301, 1992, and Vogelmeier et al., J. Appl.Physiol. 69:1843-1848, 1990, and Edwards et al., Large porous particlesfor pulmonary drug delivery, Science 276: 1868-1871, 1997, and U.S. Pat.Nos. 6,254,854; RE37,053; 6,136,295; 5,985,309; 6,253,762; 6,143,277;and 6,131,566.

[0118] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0119] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0120] The active compounds may be prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

[0121] It is advantageous to formulate oral or parenteral compositionsin dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

[0122] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds that exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0123] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0124] It is understood that the specific dose level for any particularanimal subject will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

[0125] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection,inhalation, local administration (see U.S. Pat. 5,328,470) or bystereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. USA 91:3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.,retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

[0126] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0127] IV. Compositions and Kits of the Invention

[0128] The invention also provides compositions and kits used in themethods described herein. Generally, the compositions of the inventionfor use in inhibiting proteases comprise an effective amount of a fusionprotein comprising AAT and another protease inhibitor or functionallyactive portions thereof. The compositions are generally in a suitablemedium, although they can be in lyophilized form. Suitable media for invitro use include, but are not limited to, aqueous media (such as purewater or buffers). Compositions intended for in vivo use in theinhibition of proteases and/or treatment of pathological conditions ordisease will generally be provided with a pharmaceutically acceptableexcipient or carrier, and may be in various formulations, depending onthe route of administration, dosage, stability, and other factorswell-known in the art. The compositions may be any fusion protein,polynucleotide, vector, host cell, transformed cell, reaction mixtureand/or intermediate described herein, as well as any combination. Forexample, one embodiment of a composition of the present invention is afusion protein comprising AAT or a functionally active portion thereofand SLPI or a functionally active portion thereof, together with asuitable excipient or carrier for administration to a human being.

[0129] The invention also provides kits for carrying out the methods ofthe invention. Accordingly, a variety of kits are provided in suitablepackaging. The kits may be used for any one or more of the usesdescribed herein, including in vitro and in vivo uses, including:application to biological samples (e.g., tissue or organ samples,cultures, blood, plasma, serum, urine, saliva, sputum, and the like) toinhibit protease activity in the sample and thereby stabilize proteinproteins in the sample, treatment of individuals at risk for, orsuffering from, a disease or disorder associated with an imbalance ofproteases and protease inhibitors (e.g., asthma, chronic obstructivepulmonary disease, emphysema, and otitis media and otitis externa). Kitsfor in vitro methods may also include the appropriate components tofacilitate the desired reactions of the methods, for example, buffers,enzymes, substrates, cofactors, and other necessary reagents. Suchcomponents may be in lyophilized form. Kits for in vivo administrationmay also include the appropriate components to facilitate administrationby a particular route, e.g. inhalation, intravenous administration,topical administration, subcutaneous administration, intramuscularadministration, intraarticular administration, oral administration,intraocular administration and oral administration.

[0130] The kits of the invention may optionally include a set ofinstructions, generally written instructions, although electronicstorage media (e.g. magnetic diskette or optical disk) containinginstructions are also acceptable, relating to the use of components ofthe methods of the present invention for the intended proteaseinhibition, whether in vitro or in vivo. The instructions included withthe kit generally include information as to reagents (whether includedor not in the kit) necessary for practicing the methods of thepresentation invention, instructions on how to use the kit, appropriatereaction conditions and/or appropriate administration conditions, dosagewhere appropriate, stability, storage, interpretation of results,precautionary measures if appropriate, and the like.

[0131] The component(s) of the kit may be packaged in any convenient,appropriate packaging. The components may be packaged separately, or inone or multiple combinations.

[0132] One or more compositions in the kit can be provided as a drypowder, usually lyophilized, including excipients, which on dissolutionwill provide for a reagent solution having the appropriateconcentrations for performing any of the methods described herein. Eachcomponent can be packaged in separate containers or some components canbe combined in one container where cross-reactivity and shelf lifepermit.

[0133] The relative amounts of the various components in the kits can bevaried widely to provide for concentrations of the reagents, excipients,and/or other components that substantially optimize the reactions thatneed to occur to practice the methods disclosed herein and/or to furtheroptimize the protease inhibition desired.

[0134] V. Methods of Invention

[0135] Due to their protease inhibiting activity, the fusion proteins ofthe invention can be used to inhibit the activity of one or moreproteases, either in vitro or in vivo. Methods of using the proteaseinhibitor fusion proteins of the invention in vitro can be applied, forexample, to biological samples as a means to inhibit protease activityin the sample and thereby stabilize proteins in the sample. Methods ofusing the protease inhibitor fusion proteins of the invention in vivocan be applied, for example, to the treatment of individuals sufferingfrom, or at risk for, a disease or disorder associated with an imbalanceof proteases and protease inhibitors, e.g., asthma, chronic obstructivepulmonary disease, emphysema, and otitis media and otitis externa.

[0136] The invention generally provides a method for inhibiting proteaseactivity comprising contacting the protease with a fusion protein of theinvention such that the activity of the protease is inhibited. Aprotease can be contacted with the fusion protein in vitro by, forexample, adding the fusion protein to a sample (e.g., a biologicalsample) or culture (e.g., cell culture) in vitro. Nonlimiting examplesof samples include biological fluids such as blood, plasma, serum,urine, saliva, sputum and the like, tissue samples and cellularcultures. A protease can be contacted with the fusion protein in vivo inan individual by, for example, administering the fusion protein to theindividual by an appropriate route to deliver the fusion protein to thesite of protease. Nonlimiting examples of appropriate routes ofadministration include inhalation, intravenous administration, topicaladministration, subcutaneous administration, intramuscularadministration, intraarticular administration, oral administration,intraocular administration and oral administration. The inventiongenerally provides a method of treating an individual suffering from, orat risk for, a disease or disorder involving unwanted protease activitycomprising administering to the individual an effective amount of afusion protein of the invention.

[0137] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with an imbalance ofproteases and protease inhibitors. With regards to both prophylactic andtherapeutic methods of treatment, such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics. The compositions of the present invention are usefulin treating a number of diseases where more than one protease isinvolved in the disease process. In addition, some proteases destroyprotease inhibitors, e.g., metalloproteases are known to destroy alpha1-antitrypsin. Thus, a fusion protein containing two differentinhibitors may be especially potent because the inhibitors not onlyserve to inhibit proteases involved in the disease process, but alsoserve to protect each other from proteases that would otherwise destroythem.

[0138] The amount of the compositions of the present invention, as wellas the route of delivery, will depend on the recipient and the conditionbeing treated, and may be determined without undue experiment by one ofskill in the art. Specific conditions lend themselves to particularforms of administration, as discussed below, but these are exemplaryonly.

[0139] The compositions of the present invention find particular use inrespiratory diseases, such as chronic obstructive pulmonary disease(COPD), cystic fibrosis (CF), asthma, and emphysema. For treatment ofsuch diseases, a fusion protein of the invention preferably isadministered directly to the lung via inhalation therapy. A globalstrategy for protease inhibition provided by the present inventionprovides significant therapeutic benefits for the treatment of both theairway hyperresponsiveness and the chronic airways remodeling componentsof asthma, retardation of the development of pulmonary emphysema inducedby cigarette smoking, and reduction in exacerbations of CF induced by P.aeruginosa infection. Several protease inhibitors, with complementaryinhibition spectra, are included in the fusion proteins of the presentinvention. These include alpha 1-antitrypsin (AAT), tissue inhibitors ofmetalloprotease (TIMPs), and secretory leukocyte protease inhibitor(SLPI). A fusion protein of AAT and SLPI (including functionally activeportions thereof) can be used to inhibit mast cell tryptase and chymase,and neutrophil elastase, cathepsin G and kallikrein for the treatment ofasthma, and a fusion protein of AAT and a TIMP can be used to inhibitmatrix metalloproteases and neutrophil elastase and cathepsin G for thetreatment of COPD and CF. Particularly advantageous in diseases of thelungs and airways is the fact that the compositions of the invention aresubject to direct pulmonary delivery, thus directly targeting theaffected tissue, as discussed above.

[0140] The compositions of the present invention find additional use inthe treatment of dermatological diseases such as atopic dermatitis,eczema and psoriasis, in inflammatory responses to viral infection, suchas to herpes virus types I and II, and varicella zoster virus, and intreatment of infections of the ear (e.g., otitis media and otitisexterna). In these diseases, elevated levels of neutrophil elastase, andmast cell derived proteases have been identified. Similarly, in chronicbacterial infection of the middle ear (chronic otitis media), proteaseshave been identified that can inhibited by serine protease inhibitors,and by metalloprotease inhibitors. A fusion protein of AAT with a TIMPwould be particularly advantageous in this condition, leading todecreased sequelae of the inflammatory response, and inhibition ofbacterial metalloproteases. For treatment of dermatological conditions,a fusion protein of the invention preferably is administered topicallyto an individual.

[0141] The compositions of the present invention may also be useful inHIV infections, where an aspartyl protease is a major protein coded forby the viral nucleic acid, and where protease inhibitors have been foundto be especially powerful in treatment of the disease. Furthermore, hostcell serine proteases are involved in cleavage of the HIV envelope (env)gene product, and both SLPI and AAT have been shown to inhibit HIVreplication when administered individually in in vitro assay systems.Accordingly, the invention provides a method for inhibiting HIV proteaseactivity by contacting the HIV protease with a fusion protein of theinvention. Furthermore, the invention provides methods for inhibitingHIV replication in an individual, or of decreasing HIV infectivity in anindividual, or of prolonging survival of an HIV-infected individual, byadministering to the individual a fusion protein of the invention, suchas a SLPI/AAT fusion protein.

[0142] The compositions of the present invention may also be useful inthe treatment of a number of other conditions. For the treatment ofdermatitis, psoriasis, herpes infection, corneal or epidermalulceration, chronic non-healing wounds, and sepsis, administration maybe systemic, by the methods described above, or topical, using asuitable carrier. Otitis media may be treated by oral or intramuscularadministration, or by ear canal instillation. For treatment ofrheumatoid arthritis and osteoarthritis, the administration may be localor systemic, or the fusion proteins of the present invention may beinjected directly into the affected joint(s), or applied in combinationwith a penetrating agent by patch applied over the affected area. In thetreatment of periodontal disease, administration of a penetratingtreatment may be by means of gel, toothpaste, mouthwash, spray, orlozenge, in order to slow or halt the destruction of connective tissue.For treatment of tumor metastasis and tumor angiogenesis, the fusionproteins of the present invention may be delivered intraarterially in anamount sufficient to prevent the tumor-produced proteases fromdestroying surrounding connective tissue, allowing angiogenesis ormetastasis. Other conditions, such as gastric ulceration, osteoporosis,Paget's disease of bone, glomerulonephritis, scleroderma, pressureatrophy of bone or tissues, cholesteatoma, nerve cell disorders,ischemia-reperfusion injury of organs (including local sequelae ofmyocardial anoxia), malaria, Chagas disease, parasitic eye infection,viral infection (e.g. HIV, herpes), bacterial infection, Alzheimer'sdisease, hypertension, acute leukemia, dystrophic epidermolysis bullosa,and muscular dystrophy, may be treated by methods known in the art forthe treatment of each pathological condition.

[0143] Other diseases or conditions that may be treated according to themethods of the invention using a fusion protein of the invention includeviral infections, such as herpes infections, as well as inflammatoryresponses to viral infections, such as inflammatory responses to herpesinfection. The fusion proteins of the invention also can be used in thetreatment of inflammation in general, whether resulting from a viralinfection or other cause.

[0144] For the treatment of conditions in which an excess of proteaseinhibitors is implicated, such as corneal or diabetic ulcers, or lesionsproduced by infectious microorganisms, antisense molecules to theprotease inhibitors may be administered directly to the site of thelesion by means of irrigation, salves, or other appropriate means.

[0145] The compositions of the present invention are useful in vitro, inany application where broad spectrum protection against proteases isdesired, for example, during preparation and analysis of biologicalsamples or during protein purification from tissue sources. In these andother procedures a number of proteases are released and/or activated,and a broad-spectrum protease inhibitor such as the fusion proteins ofthe present invention can be used to prevent the proteolysis of theprotein of interest in the mixture. Because the specificity of thefusion proteins can be tailored to proteases alone, and to particularclasses of proteases, it is possible to specifically inhibit proteolysisby tissue- or organ-specific proteases without affecting other proteinsof interest. In addition, the components of the fusion proteins of thepresent invention can serve to protect each other from proteolyticdigestion, thus multiplying the duration of effectiveness for theinhibitors.

[0146] This invention is further illustrated by the following examples,which should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference in their entirety.

EXAMPLES Example 1 Construction of SLPI/AAT and TIMP-1/AAT FusionProteins

[0147] A fusion protein comprising amino acids 1-107 of human SLPI fusedto amino acids 1-394 of human AAT was constructed and referred to asSLAPI. The nucleotide sequence which was used in the construction of theSLAPI fusion protein is shown in SEQ ID NO: 7 (see Table 15), in whichnucleotides 1-6 represent an XbaI restriction site, nucleotides 6-8represent a ribosome binding site, nucleotides 9-11 represent aninitiation codon, nucleotides 12-332 represent the SLPI coding sequence,nucleotides 333-335 represent a linking codon encoding a linkingmethionine residue, nucleotides 336-1517 represent the AAT codingsequence, nucleotides 1518-1520 represent a stop codon, and nucleotides1520-1525 represent a SalI restriction site. The amino acid sequence ofthe SLAPI fusion protein is shown in SEQ ID NO: 8 (see Table 16), inwhich amino acid 1 represents an initiator methionine residue, aminoacids 2-108 correspond to amino acids 1-107 of human SLPI, amino acid109 represents a linker methionine residue and amino acids 110-503correspond to amino acids 1-394 of human AAT. TABLE 15 DNA sequence usedin the construction of SLAPI tctagaccat gtctggaaag tctttcaagg ccggtgtttg60 tccaccaaag aagtccgctc aatgtttgag atacaagaag ccagaatgtc aatccgactg 120gcaatgtcca ggtaagaaga gatgttgtcc agacacttgt ggtatcaagt gtctagaccc 180agttgacacc ccaaacccaa ctagaagaaa gccaggtaag tgtccagtta cttacggtca 240atgtttgatg ttgaacccac caaacttctg tgaaatggac ggtcaatgta agagagactt 300gaagtgttgt atgggtatgt gtggtaagtc ctgtgtttcc ccagtcaagg ccatggaaga 360ccctcaaggc gacgccgctc aaaaaaccga caccagtcat cacgaccaag accatccgac 420ttttaataaa attactccaa atttagccga atttgctttt tctttgtata gacaattagc 480tcatcaaagt aattctacta acattttttt tagtcctgtt tctattgcca ctgctttcgc 540catgttgagt ttaggtacta aagccgatac ccatgacgag attttagaag gtttaaactt 600taatttgacc gaaatcccag aagcccaaat tcacgagggt tttcaagagt tgttgagaac 660tttgaatcaa cctgattctc aattgcaatt aactactggt aacggtttat ttttgtctga 720aggtttaaaa ttggttgaca aattcctaga agacgtcaag aaactatatc atagtgaggc 780ttttaccgtt aattttggtg atactgagga agctaaaaag caaattaatg attatgttga 840gaaaggcacc cagggtaaga tcgttgacct agttaaagaa ttagatcgtg ataccgtctt 900cgcactagtt aactatattt ttttcaaggg taagtgggaa cgtcctttcg aggttaaaga 960tactgaagag gaagattttc atgttgatca agttactact gtcaaagttc caatgatgaa 1020aagactgggt atgttcaata ttcaacattg caaaaaatta agttcttggg tcttattaat 1080gaagtattta ggtaacgcta ctgctatttt ttttttacca gacgaaggta agcttcaaca 1140tttagagaat gagttgactc atgacattat tactaaattt ttagagaacg aggatcgtcg 1200tagcgcttct ctgcacctgc caaagttaag tatcaccggt acttacgact taaaatctgt 1260tttaggccag ttaggtatta ccaaagtttt ttctaacggt gccgatttga gtggtgttac 1320tgaagaagct ccattaaaat tgagtaaagc tgttcacaaa gccgtcttaa ctattgatga 1380aaagggtacc gaggccgccg gcgctatgtt cctggaagct attccaatga gcattccacc 1440agaagttaaa tttaataaac cattcgtttt tctgatgatc gagcagaaca ctaaaagccc 1500attgtttatg ggtaaggttg tcaacccaac tcagaagtag tcgac 1525

[0148] TABLE 16 Amino acid sequence of SLAPI Met Ser Gly Lys Ser Phe LysAla Gly Val Cys Pro Pro Lys Lys Ser 1               5                  10                  15 Ala Gln CysLeu Arg Tyr Lys Lys Pro Glu Cys Gln Ser Asp Trp Gln            20                  25                  30 Cys Pro Gly LysLys Arg Cys Cys Pro Asp Thr Cys Gly Ile Lys Cys        35              40                      45 Leu Asp Pro Val AspThr Pro Asn Pro Thr Arg Arg Lys Pro Gly Lys    50                  55                  60 Cys Pro Val Thr Tyr GlyGln Cys Leu Met Leu Asn Pro Pro Asn Phe65                  70                  75                  80 Cys GluMet Asp Gly Gln Cys Lys Arg Asp Leu Lys Cys Cys Met Gly                85                  90                  95 Met Cys GlyLys Ser Cys Val Ser Pro Val Lys Ala Met Glu Asp Pro            100                 105                 110 Gln Gly Asp AlaAla Gln Lys Thr Asp Thr Ser His His Asp Gln Asp        115                 120                 125 His Pro Thr Phe AsnLys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe    130                 135                 140 Ser Leu Tyr Arg Gln LeuAla His Gln Ser Asn Ser Thr Asn Ile Phe145                 150                 155                 160 Phe SerPro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly                165                 170                 175 Thr Lys AlaAsp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn            180                 185                 190 Leu Thr Glu IlePro Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu        195                 200                 205 Leu Arg Thr Leu AsnGln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly    210                 215                 220 Asn Gly Leu Phe Leu SerGlu Gly Leu Lys Leu Val Asp Lys Phe Leu225                 230                 235                 240 Glu AspVal Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe                245                 250                 255 Gly Asp ThrGlu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys            260                 265                 270 Gly Thr Gln GlyLys Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp        275                 280                 285 Thr Val Phe Ala LeuVal Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu    290                 295                 300 Arg Pro Phe Glu Val LysAsp Thr Glu Glu Glu Asp Phe His Val Asp305                 310                 315                 320 Gln ValThr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met Phe                325                 330                 335 Asn Ile GlnHis Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys            340                 356                 350 Tyr Leu Gly AsnAla Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys        355                 360                 365 Leu Gln His Leu GluAsn Glu Leu Thr His Asp Ile Ile Thr Lys Phe    370                 375                 380 Leu Glu Asn Glu Asp ArgArg Ser Ala Ser Leu His Leu Pro Lys Leu385                 390                 395                 400 Ser IleThr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly                405                 410                 415 Ile Thr LysVal Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu            420                 425                 430 Glu Ala Pro LeuLys Leu Ser Lys Ala Val His Lys Ala Val Leu Thr        435                 440                 445 Ile Asp Glu Lys GlyThr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala    450                 455                 460 Ile Pro Met Ser Ile ProPro Glu Val Lys Phe Asn Lys Pro Phe Val465                 470                 475                 480 Phe LeuMet Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys                485                 490                 495 Val Val AsnPro Thr Gln Lys             500

[0149] A fusion protein comprising amino acids 1-184 of human TIMP-1fused to amino acids 1-394 of human AAT was constructed and referred toas TAPI. The nucleotide sequence which was used in the construction ofthe TAPI fusion protein is shown in SEQ ID NO: 9 (see Table 17), inwhich nucleotides 1-6 represent an XbaI restriction site, nucleotides6-8 represent a ribosome binding site, nucleotides 9-11 represent aninitiation codon, nucleotides 12-563 represent the TIMP-1 codingsequence, nucleotides 564-566 represent a linking codon encoding alinking methionine residue, nucleotides 567-1748 represent the AATcoding sequence, nucleotides 1749-1751 represent a stop codon, andnucleotides 1751-1756 represent a SalI restriction site. The amino acidsequence of the TAPI fusion protein is shown in SEQ ID NO: 10 (see Table18), in which amino acid 1 represents an initiator methionine residue,amino acids 2-185 correspond to amino acids 1-184 of human TIMP-1, aminoacid 186 represents a linker methionine residue and amino acids 187-580correspond to amino acids 1-394 of human AAT. TABLE 17 DNA sequence usedin the construction of TAPI tctagaccat gtgcacctgt gtcccacccc acccacagacggccttctgc aattccgacc 60 tcgtcatcag ggccaagttc gtggggacac cagaagtcaaccagaccacc ttataccagc 120 gttatgagat caagatgacc aagatgtata aagggttccaagccttaggg gatgccgctg 180 acatccggtt cgtctacacc cccgccatgg agagtgtctgcggatacttc cacaggtccc 240 acaaccgcag cgaggagttt ctcattgctg gaaaactgcaggatggactc ttgcacatca 300 ctacctgcag tttcgtggct ccctggaaca gcctgagcttagctcagcgc cggggcttca 360 ccaagaccta cactgttggc tgtgaggaat gcacagtgtttccctgttta tccatcccct 420 gcaaactgca gagtggcact cattgcttgt ggacggaccagctcctccaa ggctctgaaa 480 agggcttcca gtcccgtcac cttgcctgcc tgcctcgggagccagggctg tgcacctggc 540 agtccctgcg gtcccagata gccatggaag accctcaaggcgacgccgct caaaaaaccg 600 acaccagtca tcacgaccaa gaccatccga cttttaataaaattactcca aatttagccg 660 aatttgcttt ttctttgtat agacaattag ctcatcaaagtaattctact aacatttttt 720 ttagtcctgt ttctattgcc actgctttcg ccatgttgagtttaggtact aaagccgata 780 cccatgacga gattttagaa ggtttaaact ttaatttgaccgaaatccca gaagcccaaa 840 ttcacgaggg ttttcaagag ttgttgagaa ctttgaatcaacctgattct caattgcaat 900 taactactgg taacggttta tttttgtctg aaggtttaaaattggttgac aaattcctag 960 aagacgtcaa gaaactatat catagtgagg cttttaccgttaattttggt gatactgagg 1020 aagctaaaaa gcaaattaat gattatgttg agaaaggcacccagggtaag atcgttgacc 1080 tagttaaaga attagatcgt gataccgtct tcgcactagttaactatatt tttttcaagg 1140 gtaagtggga acgtcctttc gaggttaaag atactgaagaggaagatttt catgttgatc 1200 aagttactac tgtcaaagtt ccaatgatga aaagactgggtatgttcaat attcaacatt 1260 gcaaaaaatt aagttcttgg gtcttattaa tgaagtatttaggtaacgct actgctattt 1320 tttttttacc agacgaaggt aagcttcaac atttagagaatgagttgact catgacatta 1380 ttactaaatt tttagagaac gaggatcgtc gtagcgcttctctgcacctg ccaaagttaa 1440 gtatcaccgg tacttacgac ttaaaatctg ttttaggccagttaggtatt accaaagttt 1500 tttctaacgg tgccgatttg agtggtgtta ctgaagaagctccattaaaa ttgagtaaag 1560 ctgttcacaa agccgtctta actattgatg aaaagggtaccgaggccgcc ggcgctatgt 1620 tcctggaagc tattccaatg agcattccac cagaagttaaatttaataaa ccattcgttt 1680 ttctgatgat cgagcagaac actaaaagcc cattgtttatgggtaaggtt gtcaacccaa 1740 ctcagaagta gtcgac 1756

[0150] TABLE 18 Amino acid sequence of TAPI Met Cys Thr Cys Val Pro ProHis Pro Gln Thr Ala Phe Cys Asn Ser1               5                   10                  15 Asp Leu ValIle Arg Ala Lys Phe Val Gly Thr Pro Glu Val Asn Gln            20                  25                  30 Thr Thr Leu TyrGln Arg Tyr Glu Ile Lys Met Thr Lys Met Tyr Lys        35                  40                  45 Gly Phe Gln Ala LeuGly Asp Ala Ala Asp Ile Arg Phe Val Tyr Thr    50                  55                  60 Pro Ala Met Glu Ser ValCys Gly Tyr Phe His Arg Ser His Asn Arg65                  70                  75                   80 Ser GluGlu Phe Leu Ile Ala Gly Lys Leu Gln Asp Gly Leu Leu His                85                  90                  95 Ile Thr ThrCys Ser Phe Val Ala Pro Trp Asn Ser Leu Ser Leu Ala            100                 105                 110 Gln Arg Arg GlyPhe Thr Lys Thr Tyr Thr Val Gly Cys Glu Glu Cys        115         120                         125 Thr Val Phe Pro CysLeu Ser Ile Pro Cys Lys Leu Gln Ser Gly Thr    130                 135                 140 His Cys Leu Trp Thr AspGln Leu Leu Gln Gly Ser Glu Lys Gly Phe145                 150                 155                 160 Gln SerArg His Leu Ala Cys Leu Pro Arg Glu Pro Gly Leu Cys Thr                165                 170                 175 Trp Gln SerLeu Arg Ser Gln Ile Ala Met Glu Asp Pro Gln Gly Asp            180                 185                 190 Ala Ala Gln LysThr Asp Thr Ser His His Asp Gln Asp His Pro Thr        195                 200                 205 Phe Asn Lys Ile ThrPro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr    210                 215                 220 Arg Gln Leu Ala His GlnSer Asn Ser Thr Asn Ile Phe Phe Ser Pro225                 230                 235                 240 Val SerIle Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala                245                 250                 255 Asp Thr HisAsp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu            260                 265                 270 Ile Pro Glu AlaGln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr        275                 280                 285 Leu Asn Gln Pro AspSer Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu    290                 295                 300 Phe Leu Ser Glu Gly LeuLys Leu Val Asp Lys Phe Leu Glu Asp Val305                 310                 315                 320 Lys LysLeu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr                325                 330                 335 Glu Glu AlaLys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln            340                 345                 350 Gly Lys Ile ValAsp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe        355                 360                 365 Ala Leu Val Asn TyrIle Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe    370                 375                 380 Glu Val Lys Asp Thr GluGlu Glu Asp Phe His Val Asp Gln Val Thr385                 390                 395                 400 Thr ValLys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln                405                 410                 415 His Cys LysLys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly            420                 425                 430 Asn Ala Thr AlaIle Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His        435                 440                 445 Leu Glu Asn Glu LeuThr His Asp Ile Ile Thr Lys Phe Leu Glu Asn    450                 455                 460 Glu Asp Arg Arg Ser AlaSer Leu His Leu Pro Lys Leu Ser Ile Thr465                 470                 475                 480 Gly ThrTyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys                485                 490                 495 Val Phe SerAsn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro            500                 505                 510 Leu Lys Leu SerLys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu        515                 520                 525 Lys Gly Thr Glu AlaAla Gly Ala Met Phe Leu Glu Ala Ile Pro Met    530                 535                 540 Ser Ile Pro Pro Glu ValLys Phe Asn Lys Pro Phe Val Phe Leu Met545                 550                 555                 560 Ile GluGln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn                565                 570                 575 Pro Thr GlnLys             580

[0151] Expression vectors were constructed as follows: pHG42, a vectorfor assembling the expression cassette for yeast expression was clonedby sequentially adding PCR cloned fragments of the Saccharomycescerevisiae ADH2 promoter and terminator and the URA3 gene intopBluescript (pB1sc, Stratagene). Briefly, the ADH2 promoter wasamplified with 5′- Xho and BamHI sites, a 3′- Xba1 site and cloned intopB1sc cut with Xho1/Xba1. The ADH2 terminator was amplified with 5′-Xba1 and Sal1 sites, a 3′-Not1 site and cloned into the ADH2promoter-containing pB1sc vector Xba1/Not1 to create pHG40. The URA3gene was amplified with 5′- BamH1 and 3′-Xho1 sites, cloned into pHG40to generate pHG42. Genes to be expressed were cloned into the Xba1/Sal1sites 5′- to 3′- and the entire cassette removed as a Not1/Xho1 fragmentfor ligation into yeast expression vectors.

[0152] pHG62 is a yeast expression vector containing the entire S.cerevisiae 2 micron sequence cloned into pB1sc. The B form of 2 micronDNA was amplified by PCR from S. cerevisiae genomic DNA in 2 fragmentsas Not1/EcoR1 and EcoR1/Xho1 fragments using the unique EcoR1 site of 2micron DNA. The entire 2 micron DNA vector was excised Not1/Xho1 forligation and transformation yeast.

[0153] Coding sequences for the fusion proteins were constructed asfollows.

[0154] SLAPI: A synthetic SLPI gene was chemically synthesized (SigmaGenosys) with yeast-preferred codons coding for the mature peptide,amino acids 1-107 and cloned into pUC19. PCR primers were designed witha 5′- Xba1 site and a 3′-Nco1 site to subclone SLPI as a fusion withAATyc2. A synthetic AAT gene (AATyc2) was chemically synthesized (SigmaGenosys) with yeast-preferred codons that encoded a methionine residue,and amino acids 1-394 of mature AAT, and cloned into pCR4TOPO. A threefragment ligation was assembled with pHG42 Xba1/Sal1 vector, DNAencoding SLPI as a Xba1 /Nco1 fragment and DNA encoding AATyc2 as aNco1/Sal fragment and cloned into E. coli to create MetSLPI/MetAATyc2pHG42. The Not1/Xho1 fragment of MetSLPI/MetAATyc2 pHG42 was cloned intopHG62 Not1/Xho1, pKC64 Not1/Xho1, or pKC65 Not1/Xho1. Other expressionvectors which were used in the construction of SLAPI and other proteaseinhibitors were pKC64 and pKC 65, which are modified versions of pHG62with the yeast LEU2 gene inserted at the novel Pst site of 2 micron DNA.A schematic diagram of the SLAPI in the pHG62 expression vector is shownin FIG. 1. A schematic diagram of the SLAPI in the pKC65 expressionvector is shown in FIG. 3.

[0155] TAPI: TIMP-1 cDNA (Docherty et al, 1985, Nature 318, 66) wascloned by PCR from human heart cDNA into pB1sc. A 5′- Xba1 site and 3′-Nco1 site were included to allow fusion of the mature peptide codons (1-184) to the AATyc2 sequence (see SLAPI above). A three fragmentligation was assembled with pHG42 Xba1/Sal1 vector, DNA encoding TIMP-1as a Xba1/Nco1 fragment and DNA encoding AATyc2 as a Nco1/Sal fragmentand cloned into E. coli to create MetTIMP/MetAATyc2 pHG42. The Not1/Xho1fragment of MetTIMP/MetAATyc2 pHG42 was cloned into pKC62 Not1/Xho1.Another expression vectors used with TAPI was pKC64 Not1/Xho1; pKC65Not1/Xho1 is also used. A schematic diagram of the TAPI in thepHG62expression vector is shown in FIG. 2.

Example 2 Construction of N-TIMP/AAT Fusion Proteins (N-series)

[0156] The amino (NH₂ or N) terminal 126 amino acids of mature TIMP-1have been demonstrated to contain the proteolytic inhibition domain.This domain has 6 cysteines and 3 disulfide bridges required for properfolding and activity whereas the full length molecule contains 12cysteines and 6 disulfide bridges. To generate an active molecule withproper disulfide linkages, the N-terminal domain of TIMP has been fusedto AAT to generate N-TAPI. rN-TAPI (reverse-N-TAPI) was constructed asdescribed in the next Example. These molecules have a methionineinitiation codon at aa 1 followed by the first 126 aa of human TIMP-1,another methionine followed by the 394 aa of mature human AAT (N-TAPI);or the reverse for rN-TAPI.

[0157] N-TAPI

[0158] N-TAPI is a fusion comprising amino acids 1-126 of human TIMP-1fused to amino acids 1-394 of human AAT. The nucleotide sequence whichwas used in the construction of the N-TAPI fusion protein is shown inSEQ ID NO: 13 (see Table 19), in which nucleotides 1-6 represent an XbaIrestriction site, nucleotides 6-8 represent a ribosome binding site,nucleotides 9-11 represent an initiation codon, nucleotides 12-389represent the TIMP coding sequence, nucleotides 390-392 represent alinking codon encoding a linking methionine residue, nucleotides393-1574 represent the AAT coding sequence, nucleotides 1575-1577represent a stop codon and nucleotides 1577-1582 represent a SalIrestriction site. TABLE 19 DNA sequence used in the construction ofN-TAPI TCTAGACCATGTGCACCTGTGTCCCACCCCACCCACAGACGGCCTTCTGCAATTCCGACC 60TCGTCATCAGGGCCAAGTTCGTGGGGACACCAGAAGTCAACCAGACCACCTTATACCAGC 120GTTATGAGATCAAGATGACCAAGATGTATAAAGGGTTCCAAGCCTTAGGGGATGCCGCTG 180ACATCCGGTTCGTCTACACCCCCGCCATGGAGAGTGTCTGCGGATACTTCCACAGGTCCC 240ACAACCGCAGCGAGGAGTTTCTCATTGCTGGAAAACTGCAGGATGGACTCTTGCACATCA 300CTACCTGCAGTTTCGTGGCTCCCTGGAACAGCCTGAGCTTAGCTCAGCGCCGGGGCTTCA 360CCAAGACGTATACTGTTGGCTGTGAGGAAATGGAAGACCCTCAAGGCGACGCCGCTCAAA 420AAACCGACACCAGTCATCACGACCAAGACCATCCGACTTTTAATAAAATTACTCCAAATT 480TAGCCGAATTTGCTTTTTCTTTGTATAGACAATTAGCTCATCAAAGTAATTCTACTAACA 540TTTTTTTTAGTCCTGTTTCTATTGCCACTGCTTTCGCCATGTTGAGTTTAGGTACTAAAG 600CCGATACCCATGACGAGATTTTAGAAGGTTTAAACTTTAATTTGACCGAAATCCCAGAAG 660CCCAAATTCACGAGGGTTTTCAAGAGTTGTTGAGAACTTTGAATCAACCTGATTCTCAAT 720TGCAATTAACTACTGGTAACGGTTTATTTTTGTCTGAAGGTTTAAAATTGGTTGACAAAT 780TCCTAGAAGACGTCAAGAAACTATATCATAGTGAGGCTTTTACCGTTAATTTTGGTGATA 840CTGAGGAAGCTAAAAAGCAAATTAATGATTATGTTGAGAAAGGCACCCAGGGTAAGATCG 900TTGACCTAGTTAAAGAATTAGATCGTGATACCGTCTTCGCACTAGTTAACTATATTTTTT 960TCAAGGGTAAGTGGGAACGTCCTTTCGAGGTTAAAGATACTGAAGAGGAAGATTTTCATG 1020TTGATCAAGTTACTACTGTCAAAGTTCCAATGATGAAAAGACTGGGTATGTTCAATATTC 1080AACATTGCAAAAAATTAAGTTCTTGGGTCTTATTAATGAAGTATTTAGGTAACGCTACTG 1140CTATTTTTTTTTTACCAGACGAAGGTAAGCTTCAACATTTAGAGAATGAGTTGACTCATG 1200ACATTATTACTAAATTTTTAGAGAACGAGGATCGTCGTAGCGCTTCTCTGCACCTGCCAA 1260AGTTAAGTATCACCGGTACTTACGACTTAAAATCTGTTTTAGGCCAGTTAGGTATTACCA 1320AAGTTTTTTCTAACGGTGCCGATTTGAGTGGTGTTACTGAAGAAGCTCCATTAAAATTGA 1380GTAAAGCTGTTCACAAAGCCGTCTTAACTATTGATGAAAAGGGTACCGAGGCCGCCGGCG 1440CTATGTTCCTGGAAGCTATTCCAATGAGCATTCCACCAGAAGTTAAATTTAATAAACCAT 1500TCGTTTTTCTGATGATCGAGCAGAACACTAAAAGCCCATTGTTTATGGGTAAGGTTGTCA 1560ACCCAACTCAGAAGTAGTCGAG 1582

[0159] Expression vectors were as for the construction of SLAPI andTAPI, using the pKC64 Not1/Xho1 expression vector. Other vectors used inthe construction of NTAPI are pHG62 and pKC65.

[0160] The coding sequences were constructed as follows. N-TIMP-1 wascloned from TIMP-1pB1sc. PCR primers were designed with a 5′- Xba1 siteand 3′-BstZ17-1 site to subclone N-TIMP-1(1-127) as a fusion withAATyc2. A portion of AAT was cloned by PCR from AATyc2pCR4TOPO by PCR.PCR primers were designed with a 5′ BstZ17-1 site and 3′ of the uniqueMfe1 site of AAT. A three fragment ligation was assembled withAATyc2pHG42 Xba1/Mfe1 vector, DNA encoding N-TIMP-1 as a Xba1/BstZ17-1fragment and DNA encoding AATyc2 as a BstZ17-1/Mfe1 fragment and clonedinto E. coli to create MetN-TIMP-1/MetAATyc2 pHG42. The Not1/Xho1fragment of MetN-TIMP-1/MetAATyc2 pHG42 was cloned into pkC64 Not1/Xho1or pHG62. The amino acid sequence of N-TAPI is shown in SEQ ID NO: 14(see Table 20), in which amino acid 1 corresponds to an initiatormethionine residue, amino acids 2-127 represent amino acids 1-126 ofhuman TIMP-1, amino acid 128 is a linking methionine, and amino acids129-522 represent amino acids 1-394 of human AAT. TABLE 20 Amino acidsequence of N-TAPI MCTCVPPHPQTAFCNSDLVIRAKFVGTPEVNQTTLYQRYEIKMTKMY 60KGFQALGDAADIR FVYTPAMESVCGYFHRSHNRSEEFLIAGKLQDGLLHITTCSFVAPWN 120SLSLAQRRGFTKT YTVGCEE-M-EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSL 180YRQLAHQSNSTNI FFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQ 238ELLRTLNQPDSQL QLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQI 298NDYVEKGTQGKIV DLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVK 358VPMMKRLGMFNIQ HCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLE 418NEDRRSASLHLPK LSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAV 478LTIDEKGTEAAGA MFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK 522

Example 3 Reverse Orientation Fusion Proteins

[0161] The r series of proteins, here rSLAPI, rTAPI, and rN-TAPI, arefusion proteins designed with AAT at the amino terminal end of theprotein with either SLPI, TIMP, or N-TIMP-1 following at the carboxylend of the protein, and a methionine inserted at the junction betweenthe two proteins. Thus, following the initiation methionine at aminoacid 1 there are the 394 amino acids of mature human AAT, anothermethionine and then 107 and 184 amino acids respectively for maturehuman SLPI and human TIMP-1, or amino acids 1-126 of N-TIMP-127.

[0162] Construction of rSLAPI

[0163] rSLAPI, a fusion of AATyc2 plus SLPI (aa 1-107), is the reverseof SLAPI. The coding regions are fused with a novel BspE1 site insertedby PCR into the first 2 aa of SLPI as follows. A 3 piece ligation isassembled in AATyc2pHG42#3 HindIII/Sal1 vector with HindIII/BspE1 AATfragment and BspE1/Sal1 SLPI fragment. The Not1/Xho1 fragment is ligatedin to the yeast vector pKC64. pHG 62 or pKC65 are also used asexpression vectors.

[0164] The DNA sequence used in the construction of r-SLAPI is shown inSEQ ID NO: 15 (Table 21), in which nucleotides 1-6 represent an XbaIrestriction site, nucleotides 6-8 represent a ribosome binding site,nucleotides 9-11 represent an initiation codon, nucleotides 12-1193represent the coding sequence for amino acids 1 -394 of AAT, nucleotides1194-1196 represent a linking codon encoding a linking methionineresidue, nucleotides 1197-1517 represent the SLPI coding sequence,nucleotides 1578-1520 represent a stop codon and nucleotides 1520-1525represent a SalI restriction site. TABLE 21 DNA sequence used in theconstruction of rSLAPI TCTAGACCATGGAAGACCCTCAAGGCGACGCCGCTCAAAAAACCG 60ACACCAGTCATCACG ACCAAGACCATCCGACTTTTAATAAAATTACTCCAAATTTAGCCG 120AATTTGCTTTTTCTT TGTATAGACAATTAGCTCATCAAAGTAATTCTACTAACATTTTTT 180TTAGTCCTGTTTCTA TTGCCACTGCTTTCGCCATGTTGAGTTTAGGTACTAAAGCCGATA 240CCCATGACGAGATTT TAGAAGGTTTAAACTTTAATTTGACCGAAATCCCAGAAGCCCAAA 300TTCACGAGGGTTTTC AAGAGTTGTTGAGAACTTTGAATCAACCTGATTCTCAATTGCAAT 360TAACTACTGGTAACG GTTTATTTTTGTCTGAAGGTTTAAAATTGGTTGACAAATTCCTAG 420AAGACGTCAAGAAAC TATATCATAGTGAGGCTTTTACCGTTAATTTTGGTGATACTGAGG 480AAGCTAAAAAGCAAA TTAATGATTATGTTGAGAAAGGCACCCAGGGTAAGATCGTTGACC 540TAGTTAAAGAATTAG ATCGTGATACCGTCTTCGCACTAGTTAACTATATTTTTTTCAAGG 600GTAAGTGGGAACGTC CTTTCGAGGTTAAAGATACTGAAGAGGAAGATTTTCATGTTGATC 660AAGTTACTACTGTCA AAGTTCCAATGATGAAAAGACTGGGTATGTTCAATATTCAACATT 720GCAAAAAATTAAGTT CTTGGGTCTTATTAATGAAGTATTTAGGTAACGCTACTGCTATTT 780TTTTTTTACCAGACG AAGGTAAGCTTCAACATTTAGAGAATGAGTTGACTCATGACATTA 840TTACTAAATTTTTAG AGAACGAGGATCGTCGTAGCGCTTCTCTGCACCTGCCAAAGTTAA 900GTATCACCGGTACTT ACGACTTAAAATCTGTTTTAGGCCAGTTAGGTATTACCAAAGTTT 960TTTCTAACGGTGCCG ATTTGAGTGGTGTTACTGAAGAAGCTCCATTAAAATTGAGTAAAG 1020CTGTTCACAAAGCCG TCTTAACTATTGATGAAAAGGGTACCGAGGCCGCCGGCGCTATGT 1080TCCTGGAAGCTATTC CAATGAGCATTCCACCAGAAGTTAAATTTAATAAACCATTCGTTT 1140TTCTGATGATCGAGC AGAACACTAAAAGCCCATTGTTTATGGGTAAGGTTGTCAACCCAA 1200CTCAGAAGATGTCCG GAAAGTCTTTCAAGGCCGGTGTTTGTCCACCAAAGAAGTCCGCTC 1260AATGTTTGAGATACA AGAAGCCAGAATGTCAATCCGACTGGCAATGTCCAGGTAAGAAGA 1320GATGTTGTCCAGACA CTTGTGGTATCAAGTGTCTAGACCCAGTTGACACCCCAAACCCAA 1380CTAGAAGAAAGCCAG GTAAGTGTCCAGTTACTTACGGTCAATGTTTGATGTTGAACCCAC 1440CAAACTTCTGTGAAA TGGACGGTCAATGTAAGAGAGACTTGAAGTGTTGTATGGGTATGT 1500GTGGTAAGTCCTGTG TTTCCCCAGTCAAGGCCTAGTCGAC 1525

[0165] The amino acid sequence for r-SLAPI is shown in SEQ ID NO: 16(Table 22), in which in which amino acid 1 corresponds to an initiatormethionine residue, amino acids 2-395 represent amino acids 1-394 ofhuman AAT, amino acid 396 is a linking methionine, and amino acids397-503 represent amino acids 1-107 of human AAT. TABLE 22 Amino acidsequence of rSLAPI MEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSN 60STNIFFSPVSIAT AFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQP 120DSQLQLTTGNGLF LSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQ 180GKIVDLVKELDRD TVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGM 240FNIQHCKKLSSWV LLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASL 300HLPKLSITGTYDL KSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTE 360AAGAMFLEAIPMS IPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK-M-SGKSFKAGV 418CPPKKSAQCLRYK KPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYG 478QCLMLNPPNFCEM DGQCKRDLKCCMGMCGKSCVSPVKA 503

[0166] Construction of rTAPI

[0167] rTAPI, a fusion of AAT plus TIMP-1 (aa 1 -184), is the reverse ofTAPI. The coding regions were fused with a novel Pml1 site inserted byPCR into the first 3 amino acids of TIMP-1 as follows. A 3 pieceligation was assembled with HindIII/Sal1 vector pB1sc, DNA encoding3′AAT as a HindIII/Pml1 fragment and DNA encoding TIMP-1 as a Pml1/Sal1fragment. The 3′ AAT/TIMP-1 fusion Hindlll/Sal1 fragment was subclonedinto AATyc2 pHG42 Hindlll/Sal1. rTAPI was expressed in the yeast vectorpKC64 as a Not1/Xho1 fragment as for r-SLAPI. A schematic diagram of therTAPI expression vector pKC64 is shown in FIG. 4. Other vectors used forrTAPI are pHG62 and pKC65.

[0168] The DNA sequence which was used in the construction of r-TAPI isshown in SEQ ID NO: 17 (Table 23), in which nucleotides 1-6 represent anXbaI restriction site, nucleotides 6-8 represent a ribosome bindingsite, nucleotides 9-11 represent an initiation codon, nucleotides12-1193 represent the coding sequence for amino acids 1-394 of humanAAT, nucleotides 1194-1196 represent a codon encoding a linkingmethionine residue, nucleotides 1197-1748 represent codons for aminoacids 1-184 of human TIMP-1, nucleotides 1749-1751 represent a stopcodon and nucleotides 1751-1756 represent a SalI restriction site. TABLE23 DNA sequence used in the construction of rTAPITCTAGACCATGGAAGACCCTCAAGGCGACGCCGCTCAAAAAACCG 60 ACACCAGTCATCACGACCAAGACCATCCGACTTTTAATAAAATTACTCCAAATTTAGCCG 120 AATTTGCTTTTTCTTTGTATAGACAATTAGCTCATCAAAGTAATTCTACTAACATTTTTT 180 TTAGTCCTGTTTCTATTGCCACTGCTTTCGCCATGTTGAGTTTAGGTACTAAAGCCGATA 240 CCCATGACGAGATTTTAGAAGGTTTAAACTTTAATTTGACCGAAATCCCAGAAGCCCAAA 300 TTCACGAGGGTTTTCAAGAGTTGTTGAGAACTTTGAATCAACCTGATTCTCAATTGCAAT 360 TAACTACTGGTAACGGTTTATTTTTGTCTGAAGGTTTAAAATTGGTTGACAAATTCCTAG 420 AAGACGTCAAGAAACTATATCATAGTGAGGCTTTTACCGTTAATTTTGGTGATACTGAGG 480 AAGCTAAAAAGCAAATTAATGATTATGTTGAGAAAGGCACCCAGGGTAAGATCGTTGACC 540 TAGTTAAAGAATTAGATCGTGATACCGTCTTCGCACTAGTTAACTATATTTTTTTCAAGG 600 GTAAGTGGGAACGTCCTTTCGAGGTTAAAGATACTGAAGAGGAAGATTTTCATGTTGATC 660 AAGTTACTACTGTCAAAGTTCCAATGATGAAAAGACTGGGTATGTTCAATATTCAACATT 720 GCAAAAAATTAAGTTCTTGGGTCTTATTAATGAAGTATTTAGGTAACGCTACTGCTATTT 780 TTTTTTTACCAGACGAAGGTAAGCTTCAACATTTAGAGAATGAGTTGACTCATGACATTA 840 TTACTAAATTTTTAGAGAACGAGGATCGTCGTAGCGCTTCTCTGCACCTGCCAAAGTTAA 900 GTATCACCGGTACTTACGACTTAAAATCTGTTTTAGGCCAGTTAGGTATTACCAAAGTTT 960 TTTCTAACGGTGCCGATTTGAGTGGTGTTACTGAAGAAGCTCCATTAAAATTGAGTAAAG 1020 CTGTTCACAAAGCCGTCTTAACTATTGATGAAAAGGGTACCGAGGCCGCCGGCGCTATGT 1080 TCCTGGAAGCTATTCCAATGAGCATTCCACCAGAAGTTAAATTTAATAAACCATTCGTTT 1140 TTCTGATGATCGAGCAGAACACTAAAAGCCCATTGTTTATGGGTAAGGTTGTCAACCCAA 1200 CTCAGAAGATGTGCACGTGTGTCCCACCCCACCCACAGACGGCCTTCTGCAATTCCGACC 1260 TCGTCATCAGGGCCAAGTTCGTGGGGACACCAGAAGTCAACCAGACCACCTTATACCAGC 1320 GTTATGAGATCAAGATGACCAAGATGTATAAAGGGTTCCAAGCCTTAGGGGATGCCGCTG 1380 ACATCCGGTTCGTCTACACCCCCGCCATGGAGAGTGTCTGCGGATACTTCCACAGGTCCC 1440 ACAACCGCAGCGAGGAGTTTCTCATTGCTGGAAAACTGCAGGATGGACTCTTGCACATCA 1500 CTACCTGCAGTTTCGTGGCTCCCTGGAACAGCCTGAGCTTAGCTCAGCGCCGGGGCTTCA 1560 CCAAGACCTACACTGTTGGCTGTGAGGAATGCACAGTGTTTCCCTGTTTATCCATCCCCT 1620 GCAAACTGCAGAGTGGCACTCATTGCTTGTGGACGGACCAGCTCCTCCAAGGCTCTGAAA 1680 AGGGCTTCCAGTCCCGTCACCTTGCCTGCCTGCCTCGGGAGCCAGGGCTGTGCACCTGGC 1740 AGTCCCTGCGGTCCCAGATAGCCTAGTCGAC 1756

[0169] The amino acid sequence for R-TAPI is shown in SEQ ID NO: 18 (seeTable 24), in which in which amino acid 1 corresponds to an initiatormethionine residue, amino acids 2-395 represent amino acids 1-394 ofhuman AAT, amino acid 396 is a linking methionine, and amino acids397-580 represent amino acids 1-184 of human TIMP-1. TABLE 24 Amino acidsequence of rTAPI MEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSN 60STNIFFSPVSIAT AFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQP 120DSQLQLTTGNGLF LSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQ 180GKIVDLVKELDRD TVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGM 240FNIQHCKKLSSWV LLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASL 300HLPKLSITGTYDL KSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTE 360AAGAMFLEAIPMS IPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK-M-CTCVPPHPQ 418TAFCNSDLVIRAK FVGTPEVNQTTLYQRYEIKMTKMYKGFQALGDAADIRFVYTPAMESV 478CGYFHRSHNRSEE FLIAGKLQDGLLHITTCSFVAPWNSLSLAQRRGFTKTYTVGCEECTV 538FPCLSIPCKLQSG THCLWTDQLLQGSEKGFQSRHLACLPREPGLCTWQSLRSQIA 580

[0170] Construction of rN-TAPI

[0171] rN-TAPI , a fusion of AAT plus N-TIMP-127 (aa 1-126 of N-TIMP,with a leading methionine), is the reverse of N-TAPI. The coding regionswere fused with a novel Pml1 site inserted by PCR into the first 3 aa ofTIMP-1 as follows. A ligation was assembled in rTAP12pHG42 Pml1/Sal1vector with Pml1/Sal1 N-TIMP1 fragment. The Not1/Xho1 fragment wasligated into the yeast vector pKC64. Other vectors used for rNTAPI arepHG62 and pKC65.

[0172] The DNA sequence which was used in the construction of rN-TAPI isshown in SEQ ID NO: 19 (see Table 25), in which nucleotides 1-6represent an XbaI restriction site, nucleotides 6-8 represent a ribosomebinding site, nucleotides 9-11 represent an initiation codon,nucleotides 12-1193 represent the coding sequence for amino acids 1-394of human AAT, nucleotides 1194-1196 represent a codon encoding a linkingmethionine residue, nucleotides 1197-1574 represent codons for aminoacids 1-126 of human TIMP-1, nucleotides 1575-1577 represent a stopcodon and nucleotides 1577-1582 represent a Sal1 restriction site. TABLE25 DNA sequence used in the construction of rN- TAPITCTAGACCATGGAAGACCCTCAAGGCGACGCCGCTCAAAAAACCG 60 ACACCAGTCATCACGACCAAGACCATCCGACTTTTAATAAAATTACTCCAAATTTAGCCG 120 AATTTGCTTTTTCTTTGTATAGACAATTAGCTCATCAAAGTAATTCTACTAACATTTTTT 180 TTAGTCCTGTTTCTATTGCCACTGCTTTCGCCATGTTGAGTTTAGGTACTAAAGCCGATA 240 CCCATGACGAGATTTTAGAAGGTTTAAACTTTAATTTGACCGAAATCCCAGAAGCCCAAA 300 TTCACGAGGGTTTTCAAGAGTTGTTGAGAACTTTGAATCAACCTGATTCTCAATTGCAAT 360 TAACTACTGGTAACGGTTTATTTTTGTCTGAAGGTTTAAAATTGGTTGACAAATTCCTAG 420 AAGACGTCAAGAAACTATATCATAGTGAGGCTTTTACCGTTAATTTTGGTGATACTGAGG 480 AAGCTAAAAAGCAAATTAATGATTATGTTGAGAAAGGCACCCAGGGTAAGATCGTTGACC 540 TAGTTAAAGAATTAGATCGTGATACCGTCTTCGCACTAGTTAACTATATTTTTTTCAAGG 600 GTAAGTGGGAACGTCCTTTCGAGGTTAAAGATACTGAAGAGGAAGATTTTCATGTTGATC 660 AAGTTACTACTGTCAAAGTTCCAATGATGAAAAGACTGGGTATGTTCAATATTCAACATT 720 GCAAAAAATTAAGTTCTTGGGTCTTATTAATGAAGTATTTAGGTAACGCTACTGCTATTT 780 TTTTTTTACCAGACGAAGGTAAGCTTCAACATTTAGAGAATGAGTTGACTCATGACATTA 840 TTACTAAATTTTTAGAGAACGAGGATCGTCGTAGCGCTTCTCTGCACCTGCCAAAGTTAA 900 GTATCACCGGTACTTACGACTTAAAATCTGTTTTAGGCCAGTTAGGTATTACCAAAGTTT 960 TTTCTAACGGTGCCGATTTGAGTGGTGTTACTGAAGAAGCTCCATTAAAATTGAGTAAAG 1020 CTGTTCACAAAGCCGTCTTAACTATTGATGAAAAGGGTACCGAGGCCGCCGGCGCTATGT 1080 TCCTGGAAGCTATTCCAATGAGCATTCCACCAGAAGTTAAATTTAATAAACCATTCGTTT 1140 TTCTGATGATCGAGCAGAACACTAAAAGCCCATTGTTTATGGGTAAGGTTGTCAACCCAA 1200 CTCAGAAGATGTGCACGTGTGTCCCACCCCACCCACAGACGGCCTTCTGCAATTCCGACC 1260 TCGTCATCAGGGCCAAGTTCGTGGGGACACCAGAAGTCAACCAGACCACCTTATACCAGC 1320 GTTATGAGATCAAGATGACCAAGATGTATAAAGGGTTCCAAGCCTTAGGGGATGCCGCTG 1380 ACATCCGGTTCGTCTACACCCCCGCCATGGAGAGTGTCTGCGGATACTTCCACAGGTCCC 1440 ACAACCGCAGCGAGGAGTTTCTCATTGCTGGAAAACTGCAGGATGGACTCTTGCACATCA 1500 CTACCTGCAGTTTCGTGGCTCCCTGGAACAGCCTGAGCTTAGCTCAGCGCCGGGGCTTCA 1560 CCAAGACCTACACTGTTGGCTGTGAGGAATAGTCGAC 1582

[0173] The amino acid sequence for RN-TAPI is shown in SEQ ID NO: 20(see Table 26), in which amino acid 1 corresponds to an initiatormethionine residue, amino acids 2-395 represent amino acids 1-394 ofhuman AAT, amino acid 396 is a linking methionine, and amino acids397-522 represent amino acids 1-126 of human TIMP-1. TABLE 26 Amino acidsequence of rN-TAPI MEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSN 60STNIFFSPVSIAT AFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQP 120DSQLQLTTGNGLF LSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQ 180GKIVDLVKELDRD TVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGM 240FNIQHCKKLSSWV LLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASL 300HLPKLSITGTYDL KSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTE 360AAGAMFLEAIPMS IPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK-M-CTCVPPHPQ 418TAFCNSDLVIRAK FVGTPEVNQTTLYQRYEIKMTKMYKGFQALGDAADIRFVYTPAMESV 478CGYFHRSHNRSEE FLIAGKLQDGLLHITTCSFVAPWNSLSLAQRRGFTKTYTVGCEE 522

Example 4 S-Linked TAPI

[0174] Another method of creating the fusion protein is by expressingN-TIMP 1-128 (Met at aa 1 plus 127 aa of mature TIMP) which contains anadditional native cysteine at the carboxyl terminus (aa 128) which isfree to form a disulfide bridge with a free cysteine in AAT; e.g. thesingle free cysteine of AAT (at position 232, see SEQ ID NO: 2). Methodsof formation of intra- and intermolecular disulfide bonds are known inthe art. N-TIMP 1-127, which lacks C-terminal cysteine of N-TIMP 1-128,was constructed as well. N-TIMP 1-127 serves as a positive control inassays with N-TIMP 1-128. Both N-TIMP 1-127 and N-TIMP 1-128 are usefulin fusion proteins with other protease inhibitors besides AAT. N-TIMP1-128 is useful because of its terminal cysteine, allowing reaction atthe thiol group for, e.g., disulfide bridge formation with anotherpeptide chain.

[0175] N-TIMP 1-127

[0176] N-TIMP 1-127 was assembled as 5′ Xba1 and 3′ Sal1 PCR fragmentsfrom TIMP-1pBlsc in pHG42 Xba/Sal vector. The DNA sequence used in theconstruction of N-TIMP 1-127 is shown in SEQ ID NO: 21 (Table 27), inwhich nucleotides 1-6 represent an Xba1 restriction site, nucleotides6-8 represent a ribosome binding site, nucleotides 9-11 represent aninitiation codon, nucleotides 12-389 represent the coding sequence foramino acids 1-126 of TIMP, nucleotides 390-392 represent a stop codonand nucleotides 392-397 represent a SalI restriction site. TABLE 27 DNAsequence used in the construction of N-TIMP 1- 127TCTAGACCATGTGCACCTGTGTCCCACCCCACCCACAGACGGCCTTC 60 TGCAATTCCGACCTCGTCATCAGGGCCAAGTTCGTGGGGACACCAGAAGTCAACCAGACC 120 ACCTTATACCAGCGTTATGAGATCAAGATGACCAAGATGTATAAAGGGTTCCAAGCCTTA 180 GGGGATGCCGCTGACATCCGGTTCGTCTACACCCCCGCCATGGAGAGTGTCTGCGGATAC 240 TTCCACAGGTCCCACAACCGCAGCGAGGAGTTTCTCATTGCTGGAAAACTGCAGGATGGA 300 CTCTTGCACATCACTACCTGCAGTTTCGTGGCTCCCTGGAACAGCCTGAGCTTAGCTCAG 360 CGCCGGGGCTTCACCAAGACGTATACTGTTGGCTGTGAGGAATAGTCGAC 397

[0177] The amino acid sequence used in constructs containing N-TIMP1-127 is shown in SEQ ID NO: 22 (see Table 28), in which amino acid 1corresponds to an initiator methionine residue, and amino acids 2-127represent amino acids 1-126 of human TIMP-1. The construct was cloned asNot1/Xho1 fragments into pKC64 Not1/Xho1 vector. Other vectors used forthis construct are pHG62 and pKC65. TABLE 28 +HZZ,1/32 Amino acidsequence of N-TIMP 1-127 MCTCVPPHPQTAFCNSDLVIRAKFVGTPEVNQTTLYQRYEIKMTKMY60 KGFQALGDAADIR FVYTPAMESVCGYFHRSHNRSEEFLIAGKLQDGLLHITTCSFVAPWN 120SLSLAQRRGFTKT YTVGCEE

[0178] N-TIMP 1-128

[0179] N-TIMP 1-128 is assembled as 5′ Xba1 and 3′ Sal1 PCR fragmentsfrom TIMP-1pBlsc in pHG42 Xba/Sal vector. The DNA sequence used in theconstruction of N-TIMP 1-128 is shown in SEQ ID NO: 23 (see Table 29),in which nucleotides 1-6 represent an XbaI restriction site, nucleotides6-8 represent a ribosome binding site, nucleotides 9-11 represent aninitiation codon, nucleotides 12-392 represent the coding sequence foramino acids 1-127 of TIMP, nucleotides 393-395 represent a stop codonand nucleotides 395-400 represent a SalI restriction site. TABLE 29N-TIMP 1-128 DNA sequenceTCTAGACCATGTGCACCTGTGTCCCACCCCACCCACAGACGGCCTTC 60 TGCAATTCCGACCTCGTCATCAGGGCCAAGTTCGTGGGGACACCAGAAGTCAACCAGACC 120 ACCTTATACCAGCGTTATGAGATCAAGATGACCAAGATGTATAAAGGGTTCCAAGCCTTA 180 GGGGATGCCGCTGACATCCGGTTCGTCTACACCCCCGCCATGGAGAGTGTCTGCGGATAC 240 TTCCACAGGTCCCACAACCGCAGCGAGGAGTTTCTCATTGCTGGAAAACTGCAGGATGGA 300 CTCTTGCACATCACTACCTGCAGTTTCGTGGCTCCCTGGAACAGCCTGAGCTTAGCTCAG 360 CGCCGGGGCTTCACCAAGACGTATACTGTTGGCTGTGAGGAATGCTAGTCGAC 400

[0180] The amino acid sequence of N-TIMP 1-128 is shown in SEQ ID NO: 24(see Table 30), in which amino acid 1 corresponds to an initiatormethionine residue, and amino acids 2-128 represent amino acids 1-127 ofhuman TIMP-1. TABLE 30 Amino acid sequence of N-TIMP 1-128MCTCVPPHPQTAFCNSDLVIRAKFVGTPEVNQTTLYQRYEIKMTKMY 60 KGFQALGDAADIRFVYTPAMESVCGYFHRSHNRSEEFLIAGKLQDGLLHITTCSFVAPWN 120 SLSLAQRRGFTKTYTVGCEEC

[0181] N-TIMP 1-128 contains an additional native cysteine at thecarboxyl terminus (aa 128) which is free to form a disulfide bridge witha cysteine of AAT; e.g., the single free cysteine of AAT (at position232, see SEQ ID NO: 2). The construct is cloned as Not1/Xho1 fragmentsinto pKC64 Not1/Xho1 vector. Other vectors used for this construct arepHG62 and pKC65. The construction of the DNA for the N-TIMP 1-128 isdescribed in Example 2, and its DNA sequence is shown in Table 29. Theamino acid sequence of N-TIMP 1-128 is shown in Table 30.

Example 5 Expression in Yeast of a Recombinant SLPI/AAT Fusion Protein

[0182] A fusion protein of SLPI and AAT (designated SLAPI, the DNA andamino acid sequence of which are shown in SEQ ID NOS: 7 and 8,respectively), prepared as described in Example 1, and AAT alone, wereexpressed intracellularly in a yeast vector containing the followingcomponents: The ADH2 promoter and terminator drive the expression of therecombinant protein and the URA3 gene provides a selectable marker forgrowth of yeast in uracil-deficient media. The yeast 2 micron sequencesare required for autonomous replication of the vector in yeast. Yeasttransformants harboring these plasmids were grown in YEPD medium andcell lysates were analyzed for total protein, AAT concentration bycapture ELISA, and elastase inhibitory activity.

[0183] Table 31, below, shows the results of AAT and SLAPI culturesgrown in 2 YEPD-based media, Difco and Red Star. Assuming the ELISA iscapable of quantitating the AAT alone or as a fusion partner, thespecific activity, units of anti-elastase activity per mg AAT/SLAPI,allows a direct comparison of proteins expressed at different levels ina crude lysate. TABLE 31 Anti-elastase activities of AAT and SLAPI AATProtein concentra- Specific concentra- Activity, Activity, tion,activity, Protein/ tion, mg/ml units/ml units/mg ELISA, mg units/mgMedium lysate lysate protein AAT/ml AAT AAT/ 4.69 0.459 0.098 0.7140.643 Difco SLAPI/ 5.42 0.208 0.038 0.161 1.292 Difco AAT/ 3.62 0.2450.068 0.315 0.778 Red Star SLAPI/ 5.63 0.191 0.034 0.130 1.469 Red Star

[0184] Both AAT and SLAPI have detectable anti-elastase activity andspecific AAT production in crude lysates. While the expression levelsvaried for each molecule in various media, the specific activity forSLAPI was shown to be 2.0- and 1.9-fold higher than AAT in Difco and RedStar medium, respectively. These results show that the SLAPI fusionprotein has double the specific activity of AAT, indicating that theSLPI and AAT halves of the molecule each exhibit inhibitory activityagainst elastase.

Example 6 Assay for AAT Activity Using Porcine Pancreatic Elastase

[0185] Definitions/Abbreviations

[0186] rAAT: recombinant alpha 1-antitrypsin, BSA: bovine serum albumin,DMSO: dimethyl sulfoxide, OD: optical density, PPE: porcine pancreaticelastase, %CV: coefficient of variation (100×standard deviation÷mean, as%).

[0187] Materials and Equipment

[0188] Multi-channel pipettor; BioHit Proline or Eppendorf, 5-100 μL.Multi-channel pipettor; BioHit Proline or Eppendorf, 50-1200 μL.Calibrated micropipettors for 5 to 1000 μL deliveries; Gilson Pipetmanor VWR. Pipet tips; Rainin GPS-250S and Rainin GPS-1000S or VWR.Disposable reagent reservoirs for multi-channelpipetting; Costar CatalogNumber 4870, non-PVC material composition, such as LabCor 730-004.Microtiter flat bottom plate; Immulon 2, Dynex Catalog Number011-010-3855, or Nunc VWR catalog number 269620. Microplate sealers,Dynex catalog number 5701, or microplate covers, Dynex catalog number24712-163. Orbital shaker; IKA Schuttler MTS 4, or gyrotory shaker Modelg2, New Brunswick Scientific. Microplate reader; Molecular DevicesSPECTRAmax 190 or SpectraMax340 or Vmax or ThermoMax. Analyticalbalance. pH meter.

[0189] Chemicals/Reagents

[0190] Tris-HCl, Electrophoresis Grade, Fisher catalog number BP153-1.Tris Base USP Grade, Ameresco catalog number T12007. Sodium Chloride,Certified ACS. BovineSerum Albumin (BSA), Fraction V, Protease Free;Golden West Biologicals, Inc., catalog number BA1060. Purified water,Millipore SuperQ or equivalent. Porcine Pancreatic Elastase, BoehringerMannheim, catalog number 100 907, 3-5 Units/mg. DMSO, Sigma catalognumber D8779. N-Suc-Ala-Ala-Val-Ala-pNA, Bachem catalog number L-1410.rAAT standard was provided by the inventors.

[0191] Procedure

[0192] AAT Activity Buffer, 50 mM Tris-Cl, 0.5 M NaCl, pH 8.0, 0.01%BSA: was prepared by dissolving 6.35 g Tris-HCl and 1.18 g Tris-base in800 mL purified water. pH was adjusted to 8.0±0.05 with 0.05 M Tris-HClor 0.05 M Tris-base. 29.22 g NaCl was added, and 0.1 g BSA, and mixeduntil dissolved. Purified water was added to 1 liter. The solution wasfiltered through a 0.2 micron pore sized filter. This solution may bestored at 2-8° C. for up to 2 months. PPE Buffer, 0.1 M Tris-Cl, pH 8.0:Was prepared by dissolving 1.37 g Tris-HCl and 0.236 g Tris-base in 80mL purified water. The pH was adjusted to 8.0±0.05 with 0.1 M Tris-HClor 0.1 M Tris-base. Purified water was added to 100 mL. The solution wasfiltered through a 0.2 micron pore sized filter. This solution may bestored at room temperature for up to 2 months. PPE Stock, 1 mg/mL: Wasprepared by dissolving 25 mg Porcine Pancreatic Elastase (PPE) in 25 mLPPE Buffer. This solution may be stored at −60 to −80° C. up to sixmonths. Do not freeze-thaw more than once. Substrate, 20 mMN-Suc-Ala-Ala-Val-Ala-pNA in DMSO: Was prepared by dissolvingN-Suc-Ala-Ala-Val-Ala-pNA in DMSO, 25 mg in 2.27 mL DMSO or 100 mg in9.08 mL DMSO. May be stored at room temperature up to two months.

[0193] Activity Assay

[0194] Prepare PPE Cocktail: Was prepared by mixing 6 μL 1 mg/mL PPEwith 12 mL AAT Activity Buffer or amount needed according to the numberof plates to be run. One 96-well microtiter plate requires 12 mL PPECocktail.

[0195] Prepare Test Samples: Samples to be tested were serially dilutedwith AAT Activity Buffer. 100 μL of each sample was added per well intriplicate. The dilutions were targeted to achieve 30-70% inhibition ofthe elastase activity. Results outside of this range were repeated.100/well of PPE cocktail was added. 100 μL/well of assaystandard/control, test sample or blank (AAT activity buffer) was added,and shaken at 250-300 rpm on an orbital shaker for 30-60 seconds to mix.Plate was covered and incubated for 15 (±1) minutes at ambienttemperature. 10 μL substrate was added to each well except substrateblanks, and shaken at 250-300 rpm on an orbital shaker for 30-60 secondsto mix. Plate was covered and incubated the plate for 60 (±5) minutes at30±2° C. The cover was removed and Optical density of each well wasdetermined within 10 minutes using a microtiter plate reader set to400-410 nm. The data was printed and saved as text file.

[0196] Calculations

[0197] PPE enzyme activity (from manufacturer's certificate of analysis,determined using N-succinyl-L-alanyl-L-analyl-L-analine-4-nitroanilideas substrate) and test sample protein concentration are required inputs.The corrected OD for each replicate control and test sample wascalculated by subtracting the average substrate blank OD. Activity foreach replicate control and test sample was calculated. Inhibition wasdetermined as follows:

[0198] Inhibition=1- (Sample OD Average uninhibited OD)

[0199] The Units PPE present in the reaction was determined:

[0200] PPE Units in reaction=elastase specific activity in Units/mg(from manufacturer's Certificate of Analysis)×Stock Concentration inmg/mL×volume stock added in mL.

[0201] For inhibition≧0.3 and≦0.7 activity was determined in Units/mL:Activity (Units/mL)=dilution factor×Inhibition×Units PPE Present÷TotalAssay Volume (mL)

[0202] Specific Activity was determined (Units/mg):

[0203] Specific Activity=(Units/mL)÷(mg/mL)

[0204] The mean specific activity was calculated, standard deviation andCV for each sample set. The mean was determined from all data in the30-70% inhibition range.

Example 7 Porcine Pancreatic Elastase Inhibition by TAPI-1 FusionProtein

[0205] The porcine pancreatic elastase inhibitory activity of the fusionprotein, TAPI-1, which was refolded from insoluble pellet materialproduced by yeast, was assayed. The protocol of Example 6 was used, withthe following modifications: a 5 ug/ml PPE solution was used and theelastase synthetic substrate N-Suc-Ala-Ala-Ala-pNA (6 mM), both of whichwere solubilized in the following assay buffer: 50 mM Tris-HCl, 500 mMNa Cl, pH 8.0, 0.01% BSA.

[0206] Conditions were as follows: 50 ul of sample or assay buffer forblank and control. 25 uL of PPE solution to samples and control or assaybuffer for blank. Plates were incubated for one half hour at 30° C.,then 100 uL of substrate was added. Samples were read immediately inmicroplate reader at 405 nm and 30° C. Inhibition was calculated as apercent of the color development in the control over a 3-5 min. timecourse, using the following equation:

[(Abs405control−Abs405sample)÷Abs405control×100%]

[0207] Three independent trials were run, with samples taken atdifferent stages of the purification process, with the results shown inTable 32. TABLE 32 Inhibition of pancreatic porcine elastase by TAPI-1at various stages of purification Sample Conc. (mg/ml) % inhibition 10.5 97.5 2 10.0 96 3 4.0 98

[0208] These results indicate that despite alteration from its nativestate due to fusion protein construction, the TAPI fusion proteinretains a high degree of elastase inhibitory activity.

Example 8 Assay for AAT Activity Using Human Neutrophil Elastase

[0209] Materials and Equipment

[0210] 1. Human Neutrophil Elastase (HNE), Lot # EH2000-2a from AthensResearch & Technology: provided as a salt-free lyophilized powdercontaining 100 μg protein; reconstituted with 200 μL 50 mM Na Acetate,pH5.5, with 150 mM NaCl; divided into 20 μL aliquots (16.9 μM) andstored in −20° C.

[0211] 2. Suc-Ala-Ala-Pro-Val-pNA (a chromogenic substrate for HNE),from Bachem: provided as a lyophilized powder containing 50 mg peptide;reconstituted with 10 mL DMSO (8.67mM); Divided into 500 μL aliquots andstored in −20° C.

[0212] 3. Activity assay buffer: 0.1 M Tris-HCl, 0.5 M NaCl, pH 7.5,stored at room temperature

[0213] 4. 96 wells Microtiter plates, Catalog No.3474 from Costar-UltraLow Cluster

[0214] 5. Multi-channelpipettor from VWRbrand, 5-50 μL or equivalent

[0215] 6. Multi-channelpipettor from VWRbrand, 50-300 μL or equivalent

[0216] 7. Microtiter Plate Reader Versa_(max) from Molecular Devices

[0217] Methods

[0218] 1. The internal temperature of the Microtiter Plate Reader wasset to 30° C. and allowed to equilibrate at this temperature. Wavelengthof the Reader was set at 405 nm.

[0219] 2. All testing was performed in triplicate. The three resultswere averaged.

[0220] 3. The HNE Standard Curve was constructed in the range of 2.5 nMto 20 nM reaction concentration.

[0221] 4. The HNE sample was diluted in activity assay buffer to 80 nM,40 nM, 20 nM, 10 nM (HNE Standards).

[0222] 5. Substrate Solution was prepared by diluting 8.67 mM originalstock to 2 mM in the activity assay buffer.

[0223] The reagents were added per well in the following sequence:

[0224] Activity assay buffer—For the plate blank, 200 μL per well of theactivity assay buffer was added. For the substrate blank, 100 μL perwell of the activity assay buffer was added. For the HNE StandardCurve,50 μL per well of the activity assay buffer was added. For the HNEUnknowns, 100 μL per well of the activity assay buffer was added.

[0225] Human Neutrophil Elastase—For the HNE Standard Curve, 50 μL perwell of the appropriate HNE Standard was added in descending order ofthe enzyme concentration, beginning with the 80 nM HNE Standard in thefirst row. For the HNE

[0226] Unknowns, 100 μL per well of the unknown concentration sample wasadded in the first row only. Serial-dilution was performed by mixing thesample with the activity assay buffer and then transferring 100 μL perwell of the mixture to the next row. Additional serial dilutions wereperformed until the unknown concentration sample exhibited activitywithin the defined range (1 nM-20 nM HNE)

[0227] The plate was incubated for a minimum of 15 minutes at 30° C. 100μL per well of the 1 mM Substrate Solution was added to all wells exceptthe plate blank. The plate was sealed and vortexed it at a setting of 5for 1 minute. The plate was placed in the Microtiter Plate Reader, andallowed to come to 30° C. over 5 minutes. The plate was read in akinetic mode, 1 reading per minute, for 15 minutes. The concentration ofthe unknown HNE sample was determined by plotting its rate of activityon the standard curve.

[0228] The inhibitory activity of SLAPI on two different HNEpreparations was assessed by the above protocol. Human neutrophilelastase, 20 nM; recombinant AAT, 10 nM; recombinant human SLPI, 10 nM;SLAPI, 10 nM; Suc-Ala-Ala-Pro-Val-pNA, 1 mM. The reagents were incubatedfor 15 min at 30 ° C. prior to adding the substrate. The results areshown in FIG. 5, in which the inhibitory activity of 10 nM recombinantAAT (rAAT), 10 nM recombinant human SLPI (rhSLPI), and a batch of SLAPIat 10 nM, run two different times (new SLAPI and old SLAPI) arecompared. These results indicate that SLAPI has enhanced proteaseinhibitory activity on a molar basis compared to either AAP or SLPIalone.

Example 9 Tryptase Activity/Inhibition Assay by RP-HPLC and Measurementof SLAPI Activity

[0229] Materials and Equipment: Human Lung Tryptase, Lot #996290 fromCortex Biochem: provided as a 540 μg/mL(17.4 μM) liquid in 10 mM MES,300 mM NaCl, 0.02 mM Heparin, pH 6.1, with 0.02% Sodium Azide as apreservative; divided it into 20 μL aliquots and stored in −20° C.;Vasoactive Intestinal Peptide, Lot #Z0203, Product #H-3775 from Bachem:provided as a lyophilized powder containing 1 mg peptide; reconstitutedit with 2.5 mL Assay Buffer (400 μg/mL or 120 μM); divided it into 100μL aliquots and stored in −20° C.; Recombinant human Secretory LeukocyteProtease Inhibitor (rhSLPI), catalog #260-PI from R&D Systems: providedas a lyophilized powder containing 100 μg protein; reconstituted in 100mM Tris-HCl, 10 mM CaCl₂, 0.1% HSA, pH 7.5; stored as 30 μL, 8.55 μMaliquots in −80° C.; Assay buffer: 0.1M Tris-HCl, 1.0 μg/mL heparin,0.02% Triton X100, pH8.0; stored at room temperature; Trifluoroaceticacid; Acetonitrile, HPLC grade; Water, HPLC grade; Methanol, HPLC grade,100%; Equivalent substitutions for the following may be used: WatersModel 2690 Separations Module, Waters Model 996 Photo Diode ArrayDetector, Vydac 238MS54 C-18 Reversed Phase Column, 0.45×25 cm, 300 Å, 5μm, Vydac, Vydac 238GK54MS Guard Column/Cartridge System, 4.6 mmdiameter, 5 μm, Vydac, Upchurch Model A-315 Pre-column Filter, UpchurchModel A-103X Pre-column Filter Frit, Waters Screw neck vial, 12×32 mm,Part No. 186000307, Waters 30011 μL Mandrel Point Insert with PolyString, Part No. WAT094170

[0230] Methods: Mobile phases were prepared as follows: Mobile Phase A:0.1% TFA in water. 1 mLTFA was added to 999 mL HPLC grade water. MobilePhase B: 0.1% TFA in acetonitrile. 1 mL TFA was added to 999 mLacetonitrile. Mobile Phase C: 100% methanol. Pre-column filter, guardcolumn, and analytical column were attached. The column heater was setat 45° C. The wavelength of the W detector was set at 215 nm. Themaximum pressure limit was set to 2000 psi and the minimum pressurelimit to 200 psi. The flow rate was set to 1 mL/min throughout the run.The run was initiated with 85% Mobile Phase A and 15% Mobile Phase B.The gradient was held for a linear ramp from 15% Mobile Phase B at t=0minute to 42% Mobile Phase B at t=10 minutes. The gradient was held at42% Mobile Phase B for 15 minutes. The column was washed with 100%Mobile Phase C at t=30 minutes. The gradient was held for a linear rampfrom 100% Mobile Phase C at t=30 minutes to 85% Mobile Phase A and 15%Mobile Phase B. The column was re-equilibrated for 5 minute at 15%Mobile Phase B.

[0231] VIP stock was diluted to the following concentrations in assaybuffer: 32 μM, 16 μM, 8 μM, 4 μM, 2 μM (105.6 μg/mL, 52.8 μg/mL, 26.4μg/mL, 13.2 μg/mL, 6.6 μg/mL, respectively). These were the assaystandards. All of the standards were run at a 50 μL injection volume persample run. If the linearity of the resulting curve (R² value) is ≧0.970and the purity of the VIP standards was >90%, proceeded to SamplePreparation.

[0232] Tryptase was diluted to 3 nM in assay buffer. If Tryptaseconcentration was unknown, A280 analysis was performed to get theabsorbance of the unknown solution. VIP was diluted to 39 μM in assaybuffer. SLPI was diluted to 3 μM in assay buffer. The ideal molar ratioof Tryptase and rhSLPI reaction is 1 to 1000.

[0233] The reactions were run in the following manner: 1) TryptaseActivity: Negative Control (VIP only reaction): 30 μL VIP and 60 μLassay buffer were mixed. All 90 μL of the mixture was transferred into apoint insert/screw neck vial apparatus. A 50 μL (injection volume) ofthe control was assayed. Tryptase Samples: 30 μL of 3 nM Tryptase, 30 μLof 39 μM VIP, and 30 μL assay buffer were mixed in a microcentrifugetube; the tube was incubated for 1 hour at 37° C.; the reaction wasterminated with 3% TFA. All 90 μL of the mixture was transferred into apoint insert/screw neck vial apparatus. A 50 μL (injection volume) ofthe control was assayed.

[0234] 2) Tryptase-SLPI Inhibition: Negative Control (VIP onlyreaction): 30 μL VIP and 60 μL assay buffer were mixed. All 90 μL of thesolution was transferred into a point insert/screw neck vial apparatus.A 50 μL (injection volume) of the control was assayed. Positive Control(Tryptase-VIP only reaction): 30 μL of 3 nM Tryptase, 30 μL of 39 μLVIP, and 30 μL assay buffer were mixed in microcentrifuge tube; the tubewas incubated for 1 hour at 37° C.; the reaction was terminated with 3%TFA. All 90 μL of the solution was transferred into a point insert/screwneck vial apparatus. A 50 μL (injection volume) of the control wasassayed. Inhibition Reaction Samples: 30 μL of 3 nM Tryptase and 30 μLof 3 μM rhSLPI were mixed in a microcentrifuge tube; the tube wasincubated for 30 minutes at 37° C.; 30 μL of 39 μM VIP was added to thesolution; the tube was incubated for another 1 hour at 37° C.; thereaction was terminated with 3% TFA. All 90 μL of the mixture wastransferred into a point insert/screw neck vial apparatus. A 50 μL(injection volume) of each sample was assayed.

[0235] 3) Calculations: All of the measurements were based on the peaksize of the residual full-length VIP against the calibration curve.

[0236] The Tryptase activity was calculated in % activity:

[0237] 100 X [(Residual “full-length” VIP in μg/mL from the NegativeControl run—Residual “full-length” VIP in μg/mL from the Tryptase Samplerun)÷Residual “full-length” VIP in μg/mL from the Negative Control run)]

[0238] The potency of SLPI or other Tryptase Inhibitors was calculatedin % Inhibition:

[0239] 100×[(Residual “full-length” VIP in μg/mL from the InhibitionReaction Samples—Residual “full-length” VIP in μg/mL from the PositiveControl run)÷(Residual “full-length” VIP in μg/mL from the NegativeControl run—Residual “full-length” VIP in μg/mL from the PositiveControl run)]

[0240] 4) Example Calculations:

[0241] Data:

[0242] Residual “full-length” VIP from the Negative Control run 46.05μg/mL

[0243] Residual “full-length” VIP from the Tryptase Sample run (aka.Positive Control)=4.85 g/mL

[0244] Residual “full-length” VIP from the Inhibition ReactionSamples=19.96 μg/mL

[0245] Calculations:

[0246] Tryptase activity in % activity:

[0247] 100×[(46.05 μg/mL−4.85 μg/mL)÷46.05 μg/mL]=89.5% active Tryptase

[0248] Potency of SLPI or other Tryptase Inhibitors in % Inhibition:

[0249] 100×[(19.96 μg/mL−4.85 μg/mL)÷(46.05 μg/mL−4.85 μg/mL)]=36.7%Inhibition

[0250] The tryptase inhibitory activity of SLAPI, SLPI, and AAT wereassayed by the protocol above. Concentrations in these assays: tryptase,1 nM; vasoactive intestinal peptide, 15 uM; Assay buffer, 0.1M Tris-Cl(pH 8.0), 1.5 ug/mL heparin, 0.02% Triton X-100; AAT, SLPI, or SLAPI waspresent at concentrations of 0. 500, 1000, 1500, 2000, 2500, 3000, 3500,4000, and 4500 nM.

[0251] Results are shown in FIG. 6, in which the inhibition of tryptaseby various concentrations of SLPI, AAT (recombinant AAT, rAAT) and SLAPIare compared. AAT had no tryptase-inhibiting activity, and thetryptase-inhibiting activity of SLAPI was between thetryptase-inhibiting activities of SLPI and AAT. These results indicatethat despite alteration from its native state due to fusion proteinconstruction, the SLAPI fusion protein retains a high degree of tryptaseinhibitory activity. In addition, these results, combined with theresults for SLAPI inhibition of porcine and human neutrophil elastase inthe previous Examples, illustrate the bifunctionality of SLAPI inprotease inhibition, in that it inhibits both tryptase and elastase.

Example 10 Impact of TAPI on the Pathogenesis of COPD

[0252] A/J mice are exposed to long-term cigarette smoke in the absenceand presence of Ilomastat (an MMP inhibitor) or TAPI by inhalationdevice (Aerogen). Adult mice (12 weeks of age) are exposed to twocigarettes per day, 6 days/wk for 1 week, 3 or 6 mths.

[0253] A pilot experiment is run to determine if, after a one weekexposure to aerosolized Ilomastat on a daily basis, there are detectablematrix metalloprotease (MMP) neutralizing levels of Ilomastat in thebroncho-alveolar lavage (BAL) of mice. This study consists of two groupsof five animals each. One group is administered nebulized PBS only(control) for 30 minutes and then smoke, the other group gets nebulizedIlomastat (in PBS) and then smoke. After one week BAL samples are takenfrom the mice and assessed by HPLC and MMP-9 inhibition for the presenceof Ilomastat. The samples are also assayed for MMP activity using asynthetic substrate. The results from this study help refine the doselevel given to the mice in the main study to ensure that the mid-highdoses are at least neutralizing.

[0254] Main Study: Exposure time course Group # 1 wk 3 mths 6 mth 1 NS(14) 10 10 12 2 Sm (wt, A/J, Vehicle) 10 10 12 3 Sm (Ilomastat dose 1)10 10 12 4 Sm (Ilomastat dose 2) 10 10 12 5 Sm (Ilomastat dose 3) 10 1012 (160 mice total) 6 Sm (TAPI dose 1) 10 10 12 7 Sm (TAPI dose 2) 10 1012 8 Sm (TAPI dose 3) 10 10 12 (96 mice total) Total # of mice used instudy = 256

[0255] Two groups with highest doses of Ilomastat and TAPI beginning 3mths after initiation of smoking are included.

[0256] Analysis

[0257] The right lung is used for fixation/inflation and paraffinembedding for morphometry—Lm (mean distance between alveolar walls, ameasure of lung elasticity), SA/V_(o) (surface area/volume); andInflammatory cell influx: Macrophage, T cell influx are assessed byimmunohistochemistry, and neutrophils by morphology

[0258] The Left lung is used for BAL—for drug, MMP inhibition; and forTissue MMP activity.

Example 11 Matrix Metalloprotease-9 (MMP-9) Assay

[0259] Materials and Equipment:

[0260] 1. Active MMP-9 Enzyme. Catalog #PF024-5UG from Oncogene ResearchProducts. Provided as a 5 μg/50 μL stock; dilute to 500 μl by adding 450μl of 50 mM HEPES, pH 7.0, 10 mM CaCl₂ (final concentration 10 ng/μl).Divide into 100 μl aliquots, store at −80° C.

[0261] 2. Peptide Substrate. Catalog #H7145 from Bachem. Dissolve 25 mgin 1 ml of DMF (final concentration 38 mM). Store at −20° C. in glassvial.

[0262] 3. Stock Reagents:

[0263] 0.5M HEPES, pH 7.0 in ddH₂O (10×stock)

[0264] 0.1M CaCl₂ in ddH₂O (10×stock)

[0265] 0.1M Ellman's Reagent in EtOH (100×stock) Store at 4° C.

[0266] 4. 96-well Microtiter plates. Catalog #3474 from Costar—Ultra LowCluster.

[0267] 5. Microtiter Plate Reader. Versamax from Molecular Devices Inc.

[0268] Methods:

[0269] 1. Set internal temperature of Microtiter Plate Reader to 25° C.

[0270] 2. Make up fresh Reagent Mix. For one 96-well plate make up 15ml: 2 ml HEPES stock (50 mM final), 2 ml CaCl₂ stock (10 mM final), 0.5ml Ellmans Reagent stock (2.5 mM final), 0.5 ml of substrate stock (0.85mM final), 10 ml ddH₂O

[0271] 3. Add 150 μl/well of reagent mix.

[0272] 4. Add 50 μl/well of sample. Serially diluted samples should bediluted with 50 mM HEPES, pH 7.0, 10 mM CaCl₂ (dilution buffer, DB).Initially measure a 1×(undiluted) sample before proceeding with serialdilutions. The range of serial dilutions is adjusted based on activityof the 1×samples.

[0273] 5. Abs₄₁₀ is read every 10-15 min for 3 hrs.

[0274] Controls:

[0275] no MMP-9 blank=50 μl of DB.

[0276] +MMP-9 control=5 μl (50 ng or 0.54 pmols) of MMP-9 stock+45 μlDB. This should yield 1.0 AU in 2-3 hrs.

[0277] The assay is run at 25° C. to optimize enzyme stability. pHshould be≦7.0 to minimize non-enzymatic color development.

Example 12 Matrix Metalloprotease-9 (MMP-9) and Elastase InhibitoryAssay of N-TAPI

[0278] Protein from the insoluble fraction of N-TAPI (prepared asdescribed in Example 2)-containing yeast cell lystaes was refolded bythe method of Huang et al. FEBS Letters (1996), 384; 155-161. The methodwas followed exactly with the exception that dialysis to remove urea wassubstituted by slow dilution followed by diafiltration,2-mercaptoethanol was substituted by glutathione, and the finalpurification column consisted of an anion- (Q-Sepharose) rather than acation-exchange (CM-cellulose) resin.

[0279] The refolded N-TAPI-2 as well as three column fractions from thefinal purification column were subjected to the MMP-9 inhibitory assaydescribed in Example 11. All four fractions contained 1-2 mg/ml of totalprotein, and 10 μL of sample (10-20 μg) was added to an assay mixcontaining 25 ng (0.29 pmols) of MMP-9. The four N-TAPI-2 samplesyielded 93-98% inhibition of MMP-9 activity as determined by absorbanceat 410 nm over a five hour period.

[0280] Elastase activity was also measured for this N-TAPI. The assaymethod was that of Example 6. The refolded material as well as two ofthe three column fractions displayed 98% or greater inhibition using themodified PPE assay.

[0281] These results demonstrate that the fusion protein N-TAPI retainsboth the MMP-inhibiting activity of TIMP-1 and the elastase-inhibitingactivity of SLPI. Thus, this construct is truly bifunctional.

We claim:
 1. A fusion protein comprising a first protease inhibitorcomprising alpha 1-antitrypsin or a functionally active portion thereof,and a second protease inhibitor or a functionally active portionthereof.
 2. A fusion protein comprising alpha 1-antitrypsin or afunctionally active portion thereof, and secretory leukocyte proteaseinhibitor or a functionally active portion thereof.
 3. A fusion proteincomprising alpha 1-antitrypsin or a functionally active portion thereof,and a tissue inhibitor of metalloproteases or a functionally activeportion thereof.
 4. The fusion protein of claim 2, comprising a) aminoacids from about 1 to about 394 of alpha 1-antitrypsin; and b) aminoacids from about 1 to about 107 of secretory leukocyte proteaseinhibitor.
 5. A polynucleotide encoding the fusion protein of claim 1,2, 3, or
 4. 6. An expression vector comprising the polynucleotide ofclaim
 5. 7. A host cell comprising the expression vector of claim
 6. 8.A pharmaceutical composition comprising the fusion protein of claim 1,2, 3, or 4 admixed with a pharmaceutically acceptable vehicle.
 9. Amethod of producing the fusion protein of claim 1, 2, 3, or 4, saidmethod comprising culturing a transformed host cell containing anexpression vector encoding a fusion protein under conditions appropriatefor expressing said fusion protein.
 10. The method of claim 9 furthercomprising purifying said fusion protein.
 11. The fusion protein ofclaim 1 wherein the second protease inhibitor inhibits a serineprotease.
 12. The fusion protein of claim 1, wherein the second proteaseinhibitor inhibits a metalloprotease.
 13. The fusion protein of claim 1wherein the second protease inhibitor inhibits an aspartyl protease. 14.The fusion protein of claim 1 wherein the second protease inhibitorinhibits a cysteine protease.
 15. The fusion protein of claim 3 whereinthe tissue inhibitor of metalloproteases is TIMP-1 or a functionallyactive portion thereof.
 16. The fusion protein of claim 4 wherein thecarboxy terminus of amino acids from about 1 to about 394 of alpha1-antitrypsin is linked to the amino terminus of amino acids from about1 to about 107 of secretory leukocyte protease inhibitor.
 17. The fusionprotein of claim 4 wherein the carboxy terminus of amino acids fromabout 1 to about 107 of secretory leukocyte protease inhibitor is linkedto the amino terminus of amino acids from about 1 to about 394 of alpha1-antitrypsin.
 18. The fusion protein of claim 3, comprising a) aminoacids from about 1 to about 394 of alpha 1-antitrypsin; and b) aminoacids from about 1 to about 184 of tissue inhibitor ofmetalloproteases-1.
 19. The fusion protein of claim 18 wherein thecarboxy terminus of amino acids from about 1 to about 394 of alpha1-antitrypsin is linked to the amino terminus of amino acids from about1 to about 184 of tissue inhibitor of metalloproteases-1.
 20. The fusionprotein of claim 18 wherein the carboxy terminus of amino acids fromabout 1 to about 184 of tissue inhibitor of metalloproteases-1 is linkedto the amino terminus of amino acids from about 1 to about 394 of alpha1-antitrypsin.
 21. The fusion protein of claim 3 comprising a) aminoacids from about 1 to about 394 of alpha 1-antitrypsin; and b) aminoacids from about 1 to about 126 of tissue inhibitor ofmetalloproteases-1.
 22. The fusion protein of claim 21 wherein thecarboxy terminus of amino acids from about 1 to about 394 of alpha1-antitrypsin is linked to the amino terminus of amino acids from about1 to about 126 of tissue inhibitor of metalloproteases-1.
 23. The fusionprotein of claim 21 wherein the carboxy terminus of amino acids fromabout 1 to about 126 of tissue inhibitor of metalloproteases-1 is linkedto the amino terminus of amino acids from about 1 to about 394 of alpha1-antitrypsin.
 24. A fusion protein comprising a) a polypeptidecomprising amino acids from about 1 to about 394 of alpha 1-antitrypsin;and b) a polypeptide comprising amino acids from about 1 to 127 oftissue inhibitor of metalloproteases-1, wherein the alpha 1-antitrypsinpolypeptide is covalently linked to the tissue inhibitor ofmetalloproteases-1 polypeptide through a disulfide bond between aminoacid 127 of the tissue inhibitor of metalloproteases-1 polypeptide and afree cysteine residue of the alpha 1-antitrypsin polypeptide.
 25. Thefusion protein of claim 24 wherein the free cysteine residue of thealpha 1-antitrypsin polypeptide is at position 232 in SEQ ID NO:
 2. 26.A method for inhibiting protease activity, comprising contacting theprotease with the fusion protein of claims 1, 2, 3, or
 4. 27. The methodof claim 26 wherein the protease activity is associated with a disorderselected from the group consisting of emphysema, asthma, chronicobstructive pulmonary disease, cystic fibrosis, otitis media, and otitisexterna.
 28. The method of claim 26, wherein the protease activity isassociated with HIV infection.
 29. The method of claim 26, wherein thefusion protein is contacted with the protease by administering thefusion protein to an individual having the protease.
 30. A method oftreating an individual suffering from, or at risk for, a disease ordisorder involving unwanted protease activity comprising administeringto the individual an effective amount of the fusion protein of claims 1,2, 3, or
 4. 31. The method of claim 30, wherein the individual suffersfrom emphysema.
 32. The method of claim 30, wherein the individualsuffers from asthma.
 33. The method of claim 30, wherein the individualsuffers from chronic obstructive pulmonary disease.
 34. The method ofclaim 30, wherein the individual suffers from cystic fibrosis.
 35. Themethod of claim 30, wherein the individual suffers from otitis media orotitis externa.